Melanocortin 1 receptor selective compounds

ABSTRACT

A compound of general formula (1) wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 are H or methyl, R13, R14, R15 and R16 are H or alkyl, wherein L1 and L2 are linkers selected from single bond, methyl, ethyl, wherein R19, R20 and R21 are H or —CH 2 X, NT is selected from H, hydroxyl, alkyl, aminoacid, aminoacid analogue, polypeptide and functional group, CT is selected from hydrogen, hydroxyl, alkyl, aminoacid, aminoacid analogue, polypeptide and functional group shows high selectivity and high affinity for MC1-receptors in combination with effective stimulation or inhibiton of cAMP formation in MC1-receptor expressing cells but low affinity for other subtypes of MC-receptors and may be used to treat a wide range of inflammatory conditions. Also disclosed is a DNA molecule and a corresponding vector encoding the compound, a fusion protein comprising a copy of it, a vector comprising DNA encoding the fusion protein, and a pharmaceutical composition comprising the compound.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new compounds having high affinity andselectivity for the melanocortin 1 receptor (MC1-receptor). The newcompounds selectively activate or block the MC1-receptors, which makesit possible to discriminate between the physiological effects mediatedby the MC1-receptors and other subtypes of melanocortin receptors, andto afford selective pharmacological actions upon their administration toan animal or a human. The invention also relates to methods for theirmanufacture and their pharmaceutical preparations, as well as to theiruse for the treatment of medical and veterinary conditions which can beinfluenced by the MC1-receptor.

2. Related Art

Melanocortic peptides (melanocortins) are natural peptide hormones ofanimals, in particular mammals including man, which bind to andstimulate MC-receptors. Examples of melanocortins are α-MSH, β-MSH,γ-MSH, ACTH, and their peptide fragments. α-MSH is mainly known for itsability to regulate peripheral pigmentation (Eberle 1988), whereas ACTHis known to induce steroidoneogenesis (Simpson and Waterman 1988). Themelanocortic peptides also mediate a number of other physiologicalconditions. Thus, they are reported to act as immunomodulators and toaffect one or more of motivation, learning, memory, behaviour,inflammation, body temperature, pain, perception, blood pressure, heartrate, vascular tone, brain blood flow, nerve growth, placentaldevelopment, aldosteron synthesis and release, thyroxin release,spermatogenesis, ovarian weight, prolactin and FSH secretion, uterinebleeding in women, sebum and pheromone secretion, blood glucose levels,weight homeostasis, intrauterine fetal growth as well as other eventssurrounding parturition (Garrud et al., 1974, Wiegant et al., 1979,O'Donahue et al., 1981, O'Donahue & Dorsa 1982, DeWied & Jolles 1982,Klein et al., 1985, Feng et al., 1987, Lin et al. 1987, Eberle, 1988,Gruber & Callahan 1989, DeWildt et al., 1995. Fruedman 1997).

By the use of molecular cloning, genes encoding five different subtypesof MC-receptors have been identified (Chhajlani et al. 1993, Chhajlaniand Wikberg 1992, Gantz et al. 1993a, b, Mountjoy et al. 1992). TheMC-receptors belong to the class of G-protein coupled receptors whichare all built from a single peptide forming 7 transmembrane domains. Thefive MC-receptors are termed MC1, MC2, MC3, MC4 and MC5 and they allcouple in a stimulatory fashion to CAMP. Of these the MC2-receptor isthe ACTH-receptor whereas the others constitute subtypes of melanocytestimulating hormone receptors (MSH-receptors).

The MC1-receptor is present on melanocytes and melanoma cells (Low etal. 1994, Siegrist & Eberle 1995). Recent data also indicate that theMC1-receptor is expressed in limited areas (periaqueductal gray) of ratand human brain (Xia et al. 1995), as well as in the testis (Vanetti etal. 1994). Also, very importantly recently the MC1-receptor is shown tobe present on macrophages (Star et al. 1995), neutrophils (Catania etal. 1996), glioma cells and astrocytes (Wong et al. 1997), monocytes andendothelial cells (Hartmeyer et al. 1997, and references therein).Recently the MC1-receptor mRNA was also detected in the normal mousebrain (Rajora et al. 1997a), suggesting a specific function of thisreceptor in the brain. Moreover, recent data obtained byimmunohistochemical detection techniques show that MC1-receptors arepresent in testis and ovary (Thörnvall et al. 1997). This suggests aspecific role of the MC1-receptor in reproductive physiology, as well aspossibly in reproductive pathophysiology.

The MC2-receptor is the ACTH receptor. It is present in the cortex ofthe adrenal gland. The MC3-receptor mRNA is found in distinct areas ofthe brain, as well as in placental and gut tissues (Gantz et al. 1993a,Desamaud et al. 1994, Roselli-Rehfuss et al. 1993). The MC4-receptor isubiquitous in the brain (Gantz et al. 1993b, Mountjoy et al, 1994). TheMC5-receptor is expressed in the brain, as well as in several peripheraltissues (Chhajlani et al. 1993, Gantz et al. 1994, Griffon et al. 1994,Labbé et al. 1994, Barrett et al. 1994, Fathi et al. 1995). More recentdata indicate that all the 5 cloned MC-receptors have a wider tissuedistribution (Chhajlani 1996).

The five MC-receptors show-unique affinities for the melanocorticpeptides (Schiöth et al. 1995, Schiöth et al. 1996a, b, c). Thus, theMC1-receptor shows high affinity for α-MSH, but lower affinities forβ-MSH, γ-MSH and ACTH. The MC2-receptor binds ACTH with high affinity,but it does not bind the MSH peptides. The MC3-receptor show slightlyhigher affinity for γ-MSH compared to β- and α-MSH. The MC4-receptorshows slight preference for β-MSH, over α-MSH, and a very low affinityfor γ-MSH. The MC5-receptor shows the same potency order for the MSHpeptides as the MC 1-receptor, although with much lower affinities(Schiöth et al. 1995, Schiöth et al. 1996a, b, c).

A diversity of effects is induced by natural melanocortic peptides notyet fully related to the various MC-receptor subtypes. These effectsshould be mediated by different subtypes of the MC-receptors. Mostpertinent, however, is that increasing evidence indicates theMC1-receptors play an important role in the modulation of inflammation.Thus, for example, α-MSH was shown to inhibit formation of nitric oxide(NO) in cultured murine macrophages stimulated with bacteriallipopolysaccharide and γ-interferon, an effect claimed to be caused bythe inhibition of the production of NO synthase (NOS) by the stimulationof MC1-receptors in macrophages (Star et al. 1995). As NO is believed tobe a common mediator of all forms of inflammation this indicates thatstimulation of MC1-receptors mediates the anti-inflammatory effectearlier known to be induced by MSH-peptides. α-MSH is also known toincrease the formation of interleukin 10 (IL-10) in monocytes, which isbelieved to be an important component in immunosuppressive effectsinduced by α-MSH (Bhardwaj et al. 1996).

Recent studies also show that α-MSH potently inhibits the chemotacticmigration of neutrophils (Catania et al. 1996). Moreover, neutrophilswere shown to contain MC1-receptor mRNA, which was upregulated onstimulation of the neutrophils with interferon and bacteriallipopolysaccharide (Catania et al. 1996). Thus, as neutrophil migrationconstitutes an important component in early inflammation, these resultsagain indicate the importance of the MC1-receptor as mediators ofinhibition of inflammation.

In another study the injection of α-MSH, as well as the MSH-analogue[Nle⁴,D-Phe⁷]α-MSH (NDP-MSH) was shown to inhibit the release ofcytokines IL-1 and TNF-α into the blood after intra-peritoneal injectionof lipopolysaccharide (Goninard et al. 1996). This supports theanti-inflammatory role of MSH-peptides.

Important anti-inflammatory roles of MC-receptors (presumed to be of theMC1 type) have also been implicated in the brain since α-MSH inhibitsthe production of tumour necrosis factor alpha (TNF-α) in vivo, as wellas in vitro on glioma cells; in the later case α-MSH was shown toinhibit formation of TNF-α induced by bacterial endotoxin (Wong et al.1997). In another study α-MSH given intracerebroventricularly orintraperitonally inhibited formation of central TNF-α induced by locallyadministered bacterial lipopolysaccharide (Rajora et al. 1997a). TNF-αoccurs in neurological disorders, infection and injury of the brain, andis thought to underlie pathological processes in the brain. These dataindicate an important role of MC-receptors as mediators of centralanti-inflammatory actions.

Recently α-MSH was also shown to reduce inflammation in a model forinflammatory bowel disease (Rajora et al. 1997b).

The α-MSH peptide too is ascribed an important role in cutaneousbiology. Most well known is its ability to stimulate pigment formationof the skin. α-MSH may act not only on MC-receptors located tomelanocytes but also on immunocompetent and inflammatory cells,keratinocytes, fibroblasts and endothelial cells of the skin, therebymodifying keratinocyte proliferation and differentiation, and regulateendothelial cell and fibroblast cytokine production, as well asfibroblast collagenase production. α-MSH is known to down-regulate theproduction of pro-inflammatory cytokines and accessory molecules onantigen presenting cells. In contrast suppressor factors such as IL-10are upregulated by α-MSH (Luger 1997). In vivo data show that systemicapplication of α-MSH inhibits the induction and elicitation of contacthypersensitivity and induces hapten tolerance (Luger 1997). Thus, theaccumulating evidence indicates that the stimulation of MC-receptors,presumably of the MC1-receptor subtype, mediates important negativeregulation mechanisms of cutaneous inflammation and hyper-proliferativeskin diseases (Luger 1997).

In addition to these findings, Hartmeyer et al. (1997) have shown thatα-MSH increases MC1-receptor expression in dermal microvasculatureendothelial cells and causes increased release of interleukin 8 (IL-8)from these cells. This indicates a role of MC1-receptors in the skin asmodulators of inflammation and immunity (see Hartmeyer et al. 1997).

For further reading on the anti-inflammatory role of MSH peptidesreference is made to the review by Lipton and Catania (1997).

Since 1957 MSH-receptors have been known as physiological entities.Binding sites for MSH/ACTH peptides have been identified in number ofbrain and peripheral tissues (Hnatowich et al. 1989, Tatro & Reichlin1987, Lichtensteiger et al. 1993, Tatro & Entwistle 1994). Peptidestructure activity studies of these receptors have been performed onmelanophores from lower vertebrates like Rana pipiens (frog), Anoliscarolinensis (lizard) and Xenopus laevis. Receptor studies were lateralso performed by binding on melanoma cell lines. These test systemsgave comparable results and it is now known that the data obtained withthese systems refer to the MC1-receptor (Eberle 1988).

Using such test systems it was found that replacement of L-Phe by D-Phein α-MSH provided high potency and prolonged action (Sawyer et al.,1980). Cyclic [Cys⁴, Cys¹⁰]α-MSH analogues were also synthesized; theywere found to be potent melanotrophs in skin pigmentation bioassays(Knittle et al., 1983, Sawyer et al., 1982). However, while some of theprevious known natural and synthetic peptides, as well as more recentlysynthesized ones, show high affinity for MC1-receptors, theirselectivity versus other subtypes of MC-receptors are limited (see e.g.Hol et al. 1994, Adan et al. 1994, among others). Recently, however, apeptide found by phage display screening was described which showedhigher selectivity for MC1-receptors compared to other subtypes for MCreceptors (Szardenings et al. 1997). However, for clinical and otheruses this peptide showed inferior properties due to 1) comparatively lowaffinity for MC1-receptors, 2) low agonistic effects on MC1-receptorscompared to other peptide hormones and 3) low stability due to presenceof oxidizable SH groups and instability in regard of proteolyticcleavage.

There remains a need to provide means and methods to selectivelyregulate MC1-receptors. Thereby pharmacological effects affectingprocesses and conditions related to tissues and cells expressing theMC1-receptor may be elicited. These processes and conditions comprisebut are not limited to immune responses, inflammatory processes,imunotolerance, immunomodulation, allergic processes, reproductiveprocesses, melanoma and malignant diseases related to MC1-receptorexpressing cells.

There is furthermore a need to provide chemical compounds that activateMC1-receptors selectively and with high potency; to provide chemicalcompounds which antagonize the action of other hormones and agonists onMC1-receptors selectively and with high potency; and to provide a methodfor administration of said compounds to animals including man.

DESCRIPTION OF THE INVENTION

According to the present invention are disclosed novel compounds showinghigh selectivity and high affinity for MC1-receptors in combination witheffective stimulation of CAMP formation in MC1-receptor expressingcells. On the other hand the compounds of the invention show low ornegligible affinity for other subtypes of MC-receptors.

According to the present invention are also disclosed novel compoundswhich inhibit the production of nitric oxide (NO).

According to the present invention are also disclosed novel compoundswhich are immunomodulatory and anti-inflammatory.

The compounds according to the present invention are represented by thegeneral formula (1):

wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 areselected independently from each other from H and methyl, with H beingpreferred,

-   and wherein R13, R14, R15 and R16 are selected independently from    each other from H and alkyl, in particular alkyl selected from    methyl, ethyl, propyl, isopropyl, and wherein optionally one    hydrogen in R13 and one hydrogen in R14 is exchanged for a bond    between R13 and R14, and wherein optionally one hydrogen in R15 and    one hydrogen in R16 is exchanged for a bond between R15 and R16, and    wherein L1 and L2 are linkers which are independently selected from    the group consisting of single bond, methyl, ethyl, with single bond    being preferred,-   and wherein R19, R20 and R21 are selected independently from each    other from H and —CH₂X, where X is H, alkyl, substituted alkyl,    heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,    heteroalkenyl, substituted heteroalkenyl, alkynyl, substituted    alkynyl, heteroalkynyl, substituted heteroalkynyl, cycloalkyl,    substituted cycloalkyl, cycloheteroalkyl, substituted    cycloheteroalkyl, cycloalkenyl, substituted cycloalkenyl,    cycloheteroalkenyl, substituted cycloheteroalkenyl, aryl,    substituted aryl, heteroaryl, substituted heteroaryl, functional    group,-   and wherein NT is selected from H, hydroxyl, alkyl, aminoacid,    aminoacid analogue, polypeptide and functional group, and CT is    selected from hydrogen, hydroxyl, alkyl, aminoacid, aminoacid    analogue, polypeptide and functional group, and wherein each    asymmetric center (*) is in R or S configuration.

Preferably R20 is phenyl.

Recognizing that compounds according to formula (1) represent peptides,it is furthermore understood that the compounds of the invention are notlimited to peptides according to formula (1), but include also compoundswherein one or several of the nitrogens of the peptide backbone havebeen exchanged for a carbon substituted with hydrogen, and/or whereinone or several of the oxygens of the carbonyl groups of the peptidebackbone has been exchanged for two hydrogens.

The preferred stereomeric conformation of the compound (1) of theinvention is disclosed in formula (2):

wherein R1, R2, R3. R4, R5, R6, R7, R8, R9, R10, R1 and R12 are selectedindependently from each other from H and methyl, with H being preferred,

-   and wherein R13, R14, R15 and R16 are selected independently from    each other from H and alkyl, in particular alkyl selected from    methyl, ethyl, propyl, isopropyl, and wherein optionally one    hydrogen in R13 and one hydrogen in R14 is exchanged for a bond    between R13 and R14, and wherein optionally one hydrogen in R15 and    one hydrogen in R16 is exchanged for a bond between R15 and R16,-   and wherein R19, R20 and R21 are selected independently from each    other from H and —CH₂X, where X is H, alkyl, substituted alkyl,    heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,    heteroalkenyl, substituted heteroalkenyl, alkynyl, substituted    alkynyl, heteroalkynyl, substituted heteroalkynyl, cycloalkyl,    substituted cycloalkyl, cycloheteroalkyl, substituted    cycloheteroalkyl, cycloalkenyl, substituted cycloalkenyl,    cycloheteroalkenyl, substituted cycloheteroalkenyl, aryl,    substituted aryl, heteroaryl, substituted heteroaryl, functional    group,-   and wherein NT is selected from H, hydroxyl, alkyl, aminoacid,    aminoacid analogue, polypeptide and functional group, and CT is    selected from hydrogen, hydroxyl, alkyl, aminoacid, aminoacid    analogue, polypeptide and functional group.

For the compound according to formula (2) the preferred substituent ofR20 is phenyl.

Recognizing that compounds according to formula (2) represent peptides,it is furthermore understood that the compounds of the invention are notlimited to peptides according to formula (2), but include also compoundswherein one or several of the nitrogens of the peptide backbone has beenexchanged for a carbon substituted by hydrogen, and/or wherein one orseveral of the oxygens connected to carbonyl groups of the peptidebackbone has been exchanged for two hydrogens.

According to the invention is disclosed a particularly preferredcompound (3), which in the following will be termed MS05 (SEQ ID NO:1):

The invention also includes fragments of the compounds according toformula (1), (2) and (3), as detailed in the general formula (4) inwhich five moieties A, B, C, D, E have been delimitated:

wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 areselected independently from each other from H and methyl, with H beingpreferred,

-   and wherein R13, R14, R15 and R16 are selected independently from    each other from H and alkyl, in particular alkyl selected from    methyl, ethyl, propyl, isopropyl, and wherein optionally one    hydrogen in R13 and one hydrogen in R14 is exchanged for a bond    between R13 and R14, and wherein optionally one hydrogen in R15 and    one hydrogen in R16 is exchanged for a bond between R15 and R16, and    wherein L1 and L2 are linkers which are independently selected from    the group consisting of single bond, methyl, ethyl, with single bond    being preferred,-   and wherein R19, R20 and R21 are selected independently from each    other from H and —CH₂X, where X is H, alkyl, substituted alkyl,    heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,    heteroalkenyl, substituted heteroalkenyl, alkynyl, substituted    alkynyl, heteroalkynyl, substituted heteroalkynyl, cycloalkyl,    substituted cycloalkyl, cycloheteroalkyl, substituted    cycloheteroalkyl, cycloalkenyl, substituted cycloalkenyl,    cycloheteroalkenyl, substituted cycloheteroalkenyl, aryl,    substituted aryl, heteroaryl, substituted heteroaryl, functional    group,-   and wherein NT is selected from H, hydroxyl, alkyl, aminoacid,    aminoacid analogue, polypeptide or functional group, and CT is    selected from hydrogen, hydroxyl, alkyl, aminoacid, aminoacid    analogue, polypeptide, functional group, and wherein each asymmetric    center (*) is in R or S configuration,-   and wherein moiety A is optionally exchanged for hydrogen, hydroxyl,    alkyl, aminoacid, aminoacid analogue, polypeptide, or a functional    group,-   and wherein moiety B is optionally exchanged for hydrogen, hydroxyl,    alkyl, aminoacid, aminoacid analogue, polypeptide, or a functional    group,-   and wherein optionally moiety C is exchanged for aminoacid or    aminoacid analogue,-   and wherein optionally moiety D is exchanged for aminoacid or    aminoacid analogue,-   and wherein optionally moiety E is exchanged for aminoacid or    aminoacid analogue.

In a further advantageous embodiment of the invention according toformula (4) moiety C is exchanged for aminoacid or aminoacid analogue,whereas moieties D and E are retained.

In a further advantageous embodiments of the invention according toformula (4) moiety D is exchanged for aminoacid or aminoacid analogue,whereas moieties D and E are retained.

In a still further advantageous embodiment of the invention to formula(4), as defined above, moiety E is exchanged for aminoacid or aminoacidanalogue, whereas the moieties C and D are retained.

In certain preferred embodiments of the invention one or several of R1,R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 of the compoundsaccording to formulae (1), (2) or (4) are selected to be methyl, whereasthe rest is selected to be hydrogen, the selections being made so as toprevent or decelerate breakdown by proteases and/or peptidases.Moreover, the peptide backbone of the compounds according (1), (2) and(4) may be modified by exchanging, carbon, nitrogen and oxygen atoms byother atom(s), preferably oxygen, carbon and hydrogen, respectively, soas to prevent or decelerate breakdown by proteases and/or peptidases.

In even more advantageous embodiments of the invention less than 6,preferrably less than 5, more preferred less than 4 and preferably lessthan 2, and most preferred none of the R1, R2, R3, R4, R5, R6, R7, R8,R9, R10, R11 and R12 in compounds according to formula (1), (2) and (4)are selected to be methyl.

Moreover the peptide backbone of the compounds according to formula (1),(2) and (4) may be modified by exchanging carbon, nitrogen and oxygenatoms by other atom(s) so as to prevent or decelerate breakdown byproteases or peptidases, the preferred substitute for carbon beingoxygen, for nitrogen being carbon, and for oxygen, hydrogen. In apreferred embodiment of this aspect of the invention preferably lessthan 5, more preferably less than 4, even more preferably less than 3,and most preferred less than 1 of said peptide backbone carbon, nitrogenand oxygen atoms are exchanged for oxygen, carbon or hydrogen.

In the present specification the term “peptide backbone” refers to theatoms marked by their bold characters in formulae (1), (2) and (4).

In addition R19, R20 and R21 may be selected so as to provide a modifiedcompound the digestion of which by proteases or peptidases is delayed orprevented.

Included in the invention is a compound of formula (1), (2), (3) or (4)which has been subjected to minor structural alteration by the exchangeof not more than five, preferably not more than four, more preferred notmore than three, even more preferred not more than two and mostpreferred not more than one by methyl.

The invention comprises salts of the compound of the invention by knownacids, including the trifluoroacetate, pharmaceutically acceptable saltsbeing preferred.

In a preferred embodiment of the invention a rapid breakdown of thecompound of the invention by endogenous enzymes is desired as this willprovide a medicine short half life. It is however recognized that such acompound may act as a “hit-and-run” drug, the biological action of whichextends beyond the period of time within which the presence of thecompound of the invention can be detected in the respective animal; thiseffect due to a prolonged activation of the MC1-receptor.

It is also recognized that the structural variations of the compound ofinvention, as disclosed above, in particular the proper selection ofsubstituents R19, R20, and R21, can be made so as to provide compoundswith a capacity to either selectively activate or selectively block anMC1-receptor. Typical activation of MC1-receptor by a compound of theinvention leads to stimulation of the generation of second messengerelement CAMP, as shown in Example 3, as well as biological responses andpharmacological effects related to the MC1-receptor. Activation of theMC1-receptor may also lead to receptor desensitization and/or receptordown regulation, something which in turn may lead to a decreasedcapacity of the MC1-receptor to generate biological responses and/oraccumulation of CAMP. In some embodiments of the invention the capacityto afford MC1-receptor desensitization and/or MC1-receptor downregulation is a desired effect. Moreover, in further embodiments of theinvention upregulation and/or increased expression of the MC1-receptorcan be afforded by the administration of the compound of the invention,which may also be a highly desirable effect.

On the other hand typical blockade by a compound of the invention of anMC1-receptor will prevent the induction of biological response by anendogenous or exogenous MSH peptide, a peptide analogue or aMC1-receptor stimulatory compound.

Compounds according to the invention typically showing capacity toactivate MC1-receptors are represented by (but not limited to) compoundscomplying with formula (1), (2) or (4) in which R20 is aryl, substitutedaryl, heteroaryl, substituted heteroaryl, phenyl or substituted phenyl.Compounds according to the invention typically showing capacity to blockMC1-receptors are represented by (but not limited to) compoundscomplying with formula (1), (2) or (4) in which R20 is aryl, substitutedaryl, heteroaryl, substituted heteroaryl, naphthalene, or substitutednaphthalene.

Further embodiments of the invention are directed to compoundsdecreasing the formation of interleukin 1 (IL-1), interleukin 6 (IL-6),and tumour necrosis factor α (TNF-α), to afford decreased production ofnitric oxide and downregulate the activity of nitric oxide synthase(NOS). Other embodiments of the invention are directed to compoundsstimulating the production of interleukin 8 (IL-8) and/or interleukin 10(IL-10). Yet other embodiments of the invention are directed toproducing an effect opposite to that described in regard of IL-1, IL-6,TNF-α, nitric oxide, NOS, IL-8 and IL-10.

The compounds of the invention may be manufactured by conventionalchemical techniques known in the art. They are exemplified the synthesisof MS05 described in Example 1.1.

The compounds of the invention may also be manufactured by conventionalmolecular biological techniques known in the art. In this case theDNA-sequence encoding a peptide complying with formula (1), (2), (3) or(4) is typically manufactured first. The thus obtained DNA then isincorporated into an expression vector for use in a conventionalexpression system. Suitable expression systems are also known in the artand comprise eukaryotic or prokaryotic cells. Due to this possibility ofmanufacture of the compounds of the invention using molecular biologicaltechniques, the present invention also includes DNA encoding a compoundof the invention. The DNA encoding the compound of the invention may bejoined at one or both of its 3′ and 5′-ends to other DNA. The inventiontherefore includes DNA comprising a DNA sequence encoding the compoundof the invention. The DNA encoding the compound of the invention may bepresent in a vector where it is joined with other DNA at one or both ofits 3′ and 5′-ends. The invention therefore includes a vector whichcomprises DNA encoding the compound of the invention.

The present invention also includes a fusion protein comprising one orseveral copies of the sequence of a compound of the invention. Such afusion protein is typically manufactured by use of an expression systemby utilizing the proper DNA, according to the principles describedabove, and by application of procedures well known in the art.

The invention also includes a vector comprising a DNA which encodes afusion protein comprising the amino acid sequence of one or severalcopies of the compound of the invention. According to the presentinvention DNA can be exchanged for a chemically altered non-natural DNA,said non-natural DNA being capable of essentially affording the samefunction as natural DNA with respect to peptide or protein synthesis.Moreover, the DNA according to the invention can also be exchanged forRNA. It is particularly advantageous to use RNA or a non-natural RNAwhen the non-natural DNA or RNA is administered to an animal, inparticular a human, the reason being avoidance of RNA or non-natural DNArecombining with endogenous DNA of the animal or human, thus diminishingthe risk of long term side effects.

The term “alkyl” as employed herein by itself or as part of anothergroup includes a straight or branched hydrocarbon chain of up to 18,preferably from 1 to 8 carbon atoms, such as methyl, ethyl, propyl,isopropyl, tert-butyl, butyl, pentyl, hexyl, heptyl or octyl.

The term “heteroalkyl” as employed herein by itself or as part ofanother group refers to alkyl wherein one or several carbon atoms areexchanged for heteroatom.

The term “alkenyl” as employed herein by itself or as part of anothergroup includes a straight or branched hydrocarbon chain of up to 18,preferably from 2 to 8 carbon atoms comprising one or severalcarbon—carbon double bonds, such us propenyl, butenyl, pentenyl.

The term “heteroalkenyl” as employed herein by itself or as part ofanother group refers to alkenyl wherein one or several carbon atoms areexchanged for heteroatom.

The term “alkynyl” as employed herein by itself or as part of anothergroup refers to alkyl or alkenyl containing one or several carbon—carbontriple bonds.

The term “heteroalkynyl” as employed herein by itself or as part ofanother group refers to heteroalkyl or heteroalkenyl containing one orseveral carbon—carbon triple bonds.

The term “cycloalkyl” as employed herein by itself or as part of anothergroup refers to cyclic hydrocarbons containing from 3 to 12 carbons,preferably 3 to 8 carbons, such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, which may optionally be fused with1 or 2 cycles, each cycle being independently selected from the groupconsisting of cycloalkyl, cycloheteroalkyl, cycloalkenyl,cycloheteroalkenyl, aryl and/or heteroaryl.

The term “cycloheteroalkyl” as employed herein by itself or as part ofanother group refers to cycloalkyl where one or several carbon atoms areexchanged for heteroatom.

The term “cycloalkenyl” as employed herein by itself or as part ofanother group refers to cycloalkyl containing one or severalcarbon—carbon double bonds, such as cyclopentenyl and cyclohexenyl.

The term “cycloheteroalkenyl” as employed herein by itself or as part ofanother group refers to cycloheteroalkyl where one or more bonds betweencarbons, carbon and heteroatom, or heteroatoms are double.

The term “aryl” as employed herein by itself or as part of another grouprefers to phenyl which may optionally be fused with 1 or 2 cycles whichare independently selected from cycloalkyl cycloheteroalkyl,cykloalkenyl, cycloheteroalkenyl, aryl, heteroaryl.

The term “heteroaryl” as employed herein by itself or as part of anothergroup refers to a 5- to 12-membered aromatic ring, preferably 5- to6-membered aromatic ring, which includes one or more heteroatoms, andwhich may optionally be fused with 1 or 2 cycles which are independentlyselected from cycloalkyl, cycloheteroalkyl, cykloalkenyl,cycloheteroalkenyl, aryl, heteroaryl.

The term “substituted” refers to one or more hydrogens beingsubstituted, independently of each other, by alkyl, fluorinated alkyl,alkenyl, fluorinated alkenyl, alkynyl, fluorinated alkynyl, cycloalkyl,fluorinated cycloalkyl, cycloheteroalkyl, fluorinated cycloheteroalkyl,cycloalkenyl, fluorinated cycloalkenyl, cycloheteroalkenyl, fluorinatedcycloheteroalkenyl, aryl, fluorinated aryl, heteroaryl, fluorinatedheteroaryl, functional group. Moreover, if a structure connected withthe term “substituted” is a cyclic structure fused with another cyclicstructure or other cyclic structures, the latter cyclic structure(s) mayalso be substituted.

The term “halogen” as employed herein by itself or a part of anothergroup refers to chlorine, bromine, fluorine and iodine, with chlorinebeing preferred.

The term “heteroatom” as employed herein by itself or as part of anothergroup refers to nitrogen, oxygen or sulfur, to which one or morehydrogens may be connected according to its valence, and in which in thecase of nitrogen one oxygen atom may be optionally connected to it bydonor acceptor bond, thus forming N-oxide.

The term “functional group” as employ d herein by itself or as part ofanother group refers to amino, alkylamino, dialkylamino, arylamino,arylazido, heteroarylamino, heteroarylazido, hydroxy, alkylhydroxy,fluorinated alkylhydroxy, cyano, carboxy, alkylcarboxy, arylcarboxy,halogen, nitro, hydroxylamino, acyl, fluorinated acyl, nitroso,sulfonyl, sulfinyl, thio, alkylthio, arylthio.

The term “fused” as employed herein by itself or as part of anothergroup refers to two or three cycles having one or more common atoms, thepreferred maximum number of fused cycles being three.

The term “aminoacid” as employed herein by itself or as part of anothergroup refers to alanine, arginine, asparagine, aspartic acid,p-benzoyl-phenylalanine, β-cyclohexyl-alanine, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,β-(2-naphtyl)-alanine, norleucine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine, 3,4-dichlorophenylalanine,4-fluorophenylalanine, 4-nitrophenylaianine, 2-thienylalanine,3-benzothienylalanine, 4-cyanophenylalanine, 4-iodophenylalanine,4-bromophenylalanine, 4,4′-biphenylalanine, pentafluorophenylalanine, β,β-diphenylalanine, in either D- or L-conformations,D-L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, as well as othersubstances having the following general structure (5):

in which Z is H or —CH2X, where X is H, alkyl, substituted alkyl,heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,heteroalkenyl, substituted heteroalkenyl, alkynyl, substituted alkynyl,heteroalkynyl, substituted heteroalkynyl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl,cycloalkenyl, substituted cycloalkenyl, cycloheteroalkenyl, substitutedcycloheteroalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, or functional group, NT is H, functional group or bond toanother aminoacid, and CT is functional group or bond to anotheraminoacid, the compound according to formula (5) being in either D- orL-conformation.

The term “aminoacid analogue” as employed herein by itself or as part ofanother group refers to a substance having the following generalstructure (6):

wherein A is oxygen, nitrogen or carbon to which hydrogen and/or methylis attached according to valence,

-   and wherein B is oxygen or carbon to which hydrogen and/or oxygen is    attached according to valence,-   and wherein each asymmetric centre is in either R or S    configuration,-   and wherein Z is H or —CH₂X, where X is H, alkyl, substituted alkyl,    heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,    heteroalkenyl, substituted heteroalkenyl, alkynyl, substituted    alkynyl, heteroalkynyl, substituted heteroalkynyl, cycloalkyl,    substituted cyclo alkyl, cycloheteroalkyl, substituted    cycloheteroalkyl, cycloalkenyl, substituted cycloalkenyl,    cycloheteroalkenyl, substituted cycloheteroalkenyl, aryl,    substituted aryl, heteroaryl, substituted heteroaryl, or functional    group, NT is H, functional group, bond to another aminoacid, or bond    to another aminoacid analogue, and CT is functional group, bond to    another aminoacid, or bond to another aminoacid analogue.

The compounds of the present invention have unique novel propertiesconstituting their capacity to bind preferentially to the MC1-receptoramong the five known subtypes of MC-receptors. This capacity is revealedin tests on recombinant human MC-receptors, using methods described inthe literature (see Schiöth et al., Eur. J. Pharmacol., Mol. Pharm.Sect. 1995, 288, 311 and Pharmacol. & Toxicol. 1996, 79, 161). In thesetests the capacity of the compounds to compete for the binding of[¹²⁵I]-labelled NDP-MSH ([Nle⁴, D-Phe⁷]α-MSH) to recombinant humanMC-receptor subtypes expressed in COS-1 cells was evaluated, asdescribed in Example 2. With these assays the following dissociationconstants (K_(i)s) were determined for some of the compounds of theinvention:

MC1 MC3 MC4 MC5 Compound K_(i) (nM) K_(i) (nM) K_(i) (nM) K_(i) (nM)NDP-MSH 0.11 0.39 2.3 2.9 α-MSH 0.68 52.3 2030 4990 MS05 0.761365 >>50000 >>50000 MS09 0.20 7.0 40 120

In the above table the K_(i)-values for non-labeled NDP-MSH and thenatural α-MSH-peptide is given for comparison. It is evident from theTable that MS05, which is a particularly preferred compound of theinvention, shows the same high affinity for the MC1-receptor as α-MSH,whereas it shows 1800-fold lower affinity for the MC3 receptor, and morethan 65000-fold lower affinities for the MC4 and MC5 receptors,respectively. (In the above table >>50000 is intended to mean “largerthan 50000, which means that MS05 did not show any appreciable capacityto bind to either the MC4- or MC5-receptor in concentrations up to 50000nM). MS05 thereby shows very high selectivity for MC1-receptors whencompared to its affinity for MC3, MC4 and MC5 receptors. Moreover, MS05has very high affinity for the MC1-receptor. As is evident from theTable, MS09 is another preferred compound of the invention as it showsextremely high affinity for the MC-1 receptor. These properties arecombined with an increased stability in comparison with compounds knownin the art, something which seems to be due to the lack of easilyoxidizable groups (e.g. —SH groups and —SH—CH₃ group such as in cysteineand methionine). In addition MS05 and MS09 show a higher capacity tostimulate the generation of the second messenger CAMP compared topreviously known compounds (see Example 3). The combination of the veryhigh selectivity of MS05 and MS09 with their very high selectivity andgood ability to stimulate formation of CAMP make MS05 and MS09noteworthy. In Examples 3, 5 and 7 the high capacity of MS05 and MS09 toinduce effective stimulation of CAMP is demonstrated.

Examples 4–7 show the capacity of th compounds of the invention to bindto native MSH-receptors (i.e. native MC1 receptors) in melanoma cellsand macrophages, as well as to induce stimulation of CAMP in thesecells. Example 8 demonstrates the capacity of the compounds of theinvention to inhibit nitric oxide (NO) formation in macrophages where NOproduction had be n stimulated by inflammatory agents (bacteriallipopolysaccharide and interferon gamma). The results show that thecompounds of the invention are very effective inhibitors of NO which ina further sense demonstrates that the compounds of the invention areeffective anti-inflammatory agents.

In the present specification by the term “high capacity to stimulate thesecond messenger cAMP” refers to the ability of 10 nM of a compound ofthe invention to induce at least 35%, preferably at least 45%, morepreferred at least 55%, most preferred at least 65% stimulation ofcAMP-formation in the MC1-receptor expressing cells described in Example3, in comparison to that induced by 10 nM of α-MSH in the same system,using the method described in Example. 3.

However, in the present specification a preferred method to determinewhether or not a compound (“compound” is in this context also mutatismutandis termed “test compound”) shows a “high capacity to stimulate thesecond messenger cAMP” constitutes contacting aSpecified_Concentration_A of the test compound to B16-F1 mouse melanomacells (American Type Culture Collection) for a period of between 10 to60 min, with a period of between 10–30 min being more preferred, andwith a period of 20 min being most preferred, essentially using theprocedures described in Example 5, and then determining the amount ofCAMP formed in the cells. By a “high capacity to stimulate cAMP”,according to this procedure, is defined a stimulation of cAMP amountingto at least 30%, more preferably amounting to at least 40%, somewhatmore preferably amounting to at least 50%, even somewhat more preferablymounting to at least 60%, still even more preferably amounting to atleast 70%, even still even more preferably amounting to at least 8%, andmost preferably amounting to at least 90% of that induced by contactingSpecified_Concentration_A of α-MSH to B16-F1 mouse melanoma cells, usingthe same procedure as for the test compound. By “stimulation of cAMP” isin this context intended the increased level of cAMP induced over thebasal level of cAMP, the basal level of CAMP being defined as the levelof cAMP being present in the B16-F1 cells which have been subjected tothe identical procedures as when cells were contacted with testcompound, except that no test compound or α-MSH is present during theperiod allocated for contacting. According to this procedure fordetermining the capacity of the test compound to stimulate CAMP by“Specified_Concentration_A” is intended a single concentration selectedfrom within the range 1–100 nM, with 1 nM being preferred, 100 nM evenmore preferred and 10 nM most preferred.

By cAMP is in this context intended cyclic adenosine 3′,5′-monophosphate(cyclic AMP).

As pointed out above the compounds of the invention are effectiveinhibitors of NO production. In the present specification a preferredmethod to determine whether or not a compound (“compound” is in thiscontext also mutatis mutandis termed “test compound”) is an “effectiveinhibitor of NO production” constitutes contactingSpecified_Concentration_B of the test compound to mouse RAW 264.7macrophage cells (American Type Culture Collection), the cells beingconcomitantly stimulated for their production of NO by the addition ofbetween 0–300 hg/mL of bacterial lipopolysaccharide, with 50–150 ng/mLof bacterial lipopolysaccharide being more preferred, and with 100 ng/mLof bacterial lipopolysaccharide being most preferred, and the cells alsobeing concomitantly stimulated for their production of NO by theaddition of between 0–10 units/mL of mouse recombinant interferon gamma,with 3–7 units/mL of mouse recombinant interferon gamma being morepreferred, and with 5 units/mL of mouse recombinant interferon gammabeing most preferred, for a period of 1–22 hours, with 10–20 hrs beingmore preferred, and with 16 hours being most preferred, essentiallyusing the procedures described in Example 8, and then determining theamount of nitric oxide (NO) formed, the NO being determined by using anymethod well known in the art, with the method described in Example 8,where the formation of NO is determined indirectly by measuring theamount of nitrite formed, being most preferred. By an effectiveinhibitor of NO production, according to this procedure, is defined aninhibition of NO production amounting to at least 30%, more preferablyamounting to at least 40%, somewhat more preferably amounting to atleast 50%, even somewhat more preferably amounting to at least 60%,still even more preferably amounting to at least 70%, even still evenmore preferably amounting to at least 80%, and most preferably amountingto at least 90%, of the inhibition of NO production afforded bycontacting Specified_Concentration_B of α-MSH to the mouse RAW 264.7macrophage cells, using the same procedure as for the test compound. By“inhibition of NO production” is in this context intended the reductionof NO production from the level of NO production obtained in RAW 264.7cells which have been subjected to the identical procedures as whencells were contacted with test compound, except that no test compound orα-MSH is present during the period allocated for contacting. Accordingto this procedure for determining the capacity of the test compound toinhibit NO production by “Specified_Concentration_B” is intended asingle concentration selected from within the range 0.1–100 nM, with 0.1nM being preferred, 100 nM somewhat more preferred, 1 nM even morepreferred, and 10 nM most preferred.

In addition many of the compounds of the invention, in particular manyof those complying with formula (1) and formula (2), including thecompound according to formula (4), show increased stability againstbreakdown by peptidases while retaining their high selectivity andbinding affinity for MC1-receptor. These properties are particularlyvaluable if prolonged half life in the body after systemicadministration, is desired, and for causing sustained pharmacologicalactions in a living animal, in particular a human.

In addition, the compounds of the invention do not bind to MC2-receptorsto an important extent which is of advantage.

In the present specification, “MC1/MC3-selectivity” is defined as theratio of the K_(i) of a compound for an MC3-receptor (Ki-MC3) over theK_(i) of the same compound for the MC 1-receptor (K_(i)-MC1), the K_(i)values being measured as described in Example 2, using the methoddescribed by Schiöth et al. 1995 and 1996b; hence:${{{MC1}/{MC3}} - {selectivity}} = \frac{K_{i} - {MC3}}{K_{i} - {MC1}}$

In the present specification, a “very high MC1/MC3-selectivity” of acompound of the invention refers to a MC1/MC3-selectivity of at least60, preferably at least 100, more preferred of at least 200, even morepreferred of at least 700, most preferred of at least 1000, even of atleast 1200. However, in practically all of the embodiments of thepresent invention by a “very high MC1/MC3-selectivity” suffices anMC1/MC3-selectivity of at least 10, preferably at least 20, morepreferably at least 30.

In the present specification MC1/MC4-selectivity” of a compound isdefined as the ratio of the K_(i) of the compound for an MC4 receptor(K_(i)-MC4) over the K_(i) of the same compound for the MC1-receptor(K_(i)-MC1), the K_(i) values being measured as described in Example 2by using the method described by Schiöth et al. 1995 and 1996b; hence:${{{MC1}/{MC4}} - {selectivity}} = \frac{K_{i} - {MC4}}{K_{i} - {MC1}}$

In the present specification a “very high MC1/MC4-selectivity” of acompound of the invention refers to a MC1/MC4 selectivity of at least300, preferably of at least 1000, more preferred at least 3000, evenmore preferred of at least 10000, most preferred of more than 20000 andeven more than 30000 and even 50000. However, in practically all of theembodiments of the present invention by a “very highMC1/MC4-selectivity” suffices an MC1/MC4-selectivity of at least 30,preferably at least 70, more preferably at least 150.

In the present specification “MC1/MC5-selectivity” of a compound isdefined as the ratio of the K_(i) of the compound for an MC5 receptor(K_(i)-MC5) over the K_(i) of the same compound for the MC1-receptor(K_(i)-MC1), the K_(i) values being measured as described in Example 2,using the method described by Schiöth et al. 1995 and 1996b; hence:${{{MC1}/{MC5}} - {selectivity}} = \frac{K_{i} - {MC5}}{K_{i} - {MC1}}$

In the present specification a “very high MC1/MC5-selectivity” of acompound of the invention refers to a MC1/MC5 selectivity of at least300, preferably of more than 1000, more preferred of at least 3000, evenmore preferred of at least 10000, most preferred at least 20000 and evenmore than 30000 and 50000. However, in practically all of theembodiments of the present invention by a “very highMC1/MC5-selectivity” suffices an MC1/MC5-selectivity of at least 30,preferably at least 70, more preferably at least 150.

In the present context a “very high affinity” of a compound of theinvention for an MC1-receptor indicates it exhibits a K_(i) value ofless than 30 nM, preferably of less than 15 nM, more preferred of lessthan 7 nM, even more preferred of less than 5 nM, most preferred of lessthan 3 nM, even less than 2 nM and less than 1 nM, the K_(i) value forthe MC1-receptor being measured as described in Example 2, by using themethod described by Schiöth et al. 1995 and 1996b.

However, in the present context a “very high affinity” of a compound ofthe invention for an MC1-receptor is intended that it exhibits a K_(i)value of less than 30 nM, preferably of less than 15 nM, more preferredof less than 7 nM, even more preferred of less than 5 nM, most preferredof less than 3 nM, even less than 2 nM and less than 1 nM, the K_(i)value for the MC1-receptor being measured as described in Example 4using mouse B16-F1 melanoma cells.

In the present context whenever reference is made to the use of an MC1receptor, or whenever reference is made to the human MC1 receptor, or tothe MC1 receptor described by Chhajlani & Wikberg (FEBS Lett, 1992, 309,417), or to the MC1 receptor used by Schioth et al. Eur. J. Pharmacol.Mol. Pharm Sect, 1995, 288, 311–317 or Schioth, et al., Pharmacol.Toxicol. 1996, 79, 161–165, is intended the MC1 receptor which isencoded by the DNA which is available from Deutche Sammlung vonMikroorganismen und Zellkulturen (DSM), Braunschweig, Germany under theaccession number DSM 7214, and which is described in the patentapplication no. PCT/DK93100273 (WO94/04674).

However, in the present context, whenever reference to use of an MC1receptor is made the receptor to be used can also be selected from anative MSH-receptor being present on melanoma cells of mammalian orhuman origin, without any matter of the present application beingaltered; the selection of the native MSH-receptor on B16-F1 melanomacells being a preferred choice.

In the present context whenever reference is made to an MSH-receptor isfurthermore also intended a binding site which is capable of binding[¹²⁵I] NDP-MSH with a dissociation constant of 10 nM or lower, thedissociation constant being determined by using a radio-ligand bindingprocedure well known in the art, the preferred approach being describedby Schioth et al. Eur. J. Pharmacol. Mol. Pharm Sect, 1995, 288, 311–317or Schioth, et al. Pharmacol. Toxicol. 1996, 79, 161–165, with theextension that the MSH-receptor can be present in any natural ornon-natural cell-line of mammalian or human origin. Moreover, accordingto this definition of the MSH-receptor the [¹²⁵I] NDP-MSH binding can belargely prevented by the presence of between 0.1 μM to 10 μM of α-MSHduring the assaying procedure.

In the present context whenever reference is made to mouse RAW 264.7macrophage cells, the RAW 264.7 macrophage cells can be exchanged withany other cells of mammalian or human origin that possessesMSH-receptors, and which NO production is inhibited by the addition ofα-MSH, without any matter of the present context being altered.

Particularly preferred compounds according to the invention comprised byone or several of the structural formulas (1), (2), (3), (4), and forwhich one, preferably two, more preferred three, most preferred four ofthe properties selected from the group high MC1/MC3-selectivity, highMC1/MC4-selectivity, high MC1/MC5-selectivity and high affinity for anMC1-receptor are combined. Moreover, an further preferred compound ofthe invention is one in which the said properties are combined with ahigh capacity to stimulate the second messenger cAMP.

By the term “high selectivity for MC1 receptors” is in the presentcontext intended that a compound shows three, more preferably two, andmost preferably one of the properties selected from the group of veryhigh MC1/MC3-selectivity, very high MC1/MC4-selectivity, very highMC1/MC5-selectivity.

However, a very preferred compound according to the invention iscomprised by one or several of the structural formulas (1), (2), (3),(4), which compound is also an effective inhibitor of NO production.

Marked positive treatment effects were found upon administration of MS05to BALB/C mice. Animals were sensitized by injection of2,4-dinitrofluorobenzene (DNFB). First 30 μL of 0.5% DNFB wasadministered to the shaved abdomen of the mice and after 4 days 10 μL of0.3% DNFB was challenged to one paw, another paw being unchallenged andserving as a control. MS05 was administered, using a solution of MS05 in0.9% saline, intraperitonally two hours before sensitization, and thenthe same dose was given intra-peritonally for four consecutive days,each dose of MS05 amounting to 0.05, 0.25, 0.37, 0.5 and 0.75 mg/kg.These treatments with MS05 were found to inhibit paw oedema by 3, 5, 12,39 and 10%, respectively, compared to animals that were subjected to thesame DNFB administration but which had not been given MS05. Moreover, inthese tests the DNFB induced a marked increase in total blood white cellcounts as w ll as marked increase in granulocyte counts of the blood,the increas in total white cell blood counts and granulocyte countsbeing essentially normalized by the MS05 treatment, the best effectbeing seen at 0.5 mg/kg of daily intraperitonally administrations ofMS05. The DNFB administration also increased the count oflymphocytes/monocytes in the blood and this increase was essentiallyreturned to normal levels upon the administration of MS05. Bloodplatelet counts was also increased in DNFB treated animals; the increasebeing returned towards the normal by administration of MS05. Similarreduction of the oedema induced by DNFB sensitization of the ears ofBALB/c mice was seen upon intravenous injections of either MS05 or MS09,using solutions of MS05 and MS09 in 0.9% saline, to the animals. Thus,these results show that a compound of the invention prevents,ameliorates, and/or inhibits contact hypersensitivity, sensitization bya hapten, and/or has a positive treatment effect on oedema. Moreoverthese results show that the compound of the invention is capable ofnormalizing blood cell counts. Therefore the compound of the inventionis in a further sense immunomodulatory. Moreover, these resultsdemonstrate the capacity of a compound of the invention to beadministrated in form of a pharmaceutical.

In further tests the MS05 or MS09 was administered to humanmicrovascular endothelial cells (HMEC-1 cells; Department of Health &Human Services, Centres for Disease Control and Prevention, Atlanta, Ga.30333, USA) in vitro. The HMEC-1 cells responded with an upregulatedexpression of mRNA for adhesion molecules ICAM-1, VCAM and E-Selectin,as well as by upregulation of the ICAM-1, VCAM and E-Selectin proteins,upon administration of TNF α(10 ng/mL). The administration of MS05 orMS09 (preferred concentrations of MS05 and MS09 being within 0.01 nM to10 μM, and preferred times for contacting the cells with MS05 or MS09being 3–48 hours) led to inhibition of the upregulated expression ofmRNA for adhesion molecules ICAM-1, VCAM and E-Selectin, as well as toinhibition of the upregulation of the ICAM-1, VCAM and E-Selectinproteins. These results demonstrate the capacity of the compound of theinvention to be immunomodulatory, to be useful for the treatment ofinflammation related to the vasculature, e.g. having positive treatmenteffects in vasculitis. In this context by ICAM-1 is intendedintercellular adhesion molecule type 1, by VCAM is intended vascularadhesion molecule, and by E-Selectin is intended endothelial selectin(Sluiter et al, J. Cardiovasc. Pharmacol. 1993, 22 Suppl 4: S37–44,Elangbam et al., Vet. Pathol. 1997 Jan, 34(1): 61–73).

The compounds of the invention can be used for the treatment anddiagnosis of diseases and disorders and/or pathological conditions,including preventive treatment, in an animal, in particular a mammal,but they are most preferably used for these purposes in man. Moreover,the compounds of the invention can be used also in healthy individualsand in healthy animals to change their physiological state.

In such treatment or diagnosis the compound of the invention isadministered in form of a pharmaceutical composition further comprisinga pharmaceutical acceptable carrier and, optionally, tabletting agents,wetting agents, binders and fillers, preservatives, such as antioxidantsand anti-microbial agents, buffers and salts. Preferred carrierscomprise injection media, particularly water. The compositions areadministered by any conventional route including the oral, enteral,rectal and parenteral routes. Parenteral routes comprise intravenous,intramuscular, subcutaneous and peritoneal injection. The compounds ofthe invention may also be administered by inhalation, as nasal spray,and topically on the skin. They may also be administered epidurally,intrathecally and intracerebroventricularly.

In particular the pharmaceutical composition containing apharmacologically effective amount of a compound of the invention isadministered to an animal, in particular man, for alteration ofphysiological state, diagnosis, prevention or therapeutic treatment ofdiseases, in particular conditions which are positively affected by thestimulation of MC1-receptors or alternatively positively affected byblockade, and/or by downregulation, and/or by desensitization ofMC1-receptors. Examples of such conditions include inflammation of anytype and any origin. In particular is therewith intended inflammation orany related condition as well as any condition involving the action ofmacrophages, neutrophils, monocytes, keratinocytes, fibroblasts,melanocytes, pigment cells and endothelial cells. Moreover included areconditions caused by or associated with increased production and/orrelease of inflammatory cytokines such as interleukins, in particularinterleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factorα-(TNF-α). Thus the compounds of the invention are useful forimmunomodulatory treatment in man, mammals and animals. Included arealso conditions associated with increased production of nitric oxide(NO) as well as upregulated activity of nitric oxide synthase (NOS).Moreover, the compounds of the invention are useful for treatingconditions related to the testis and ovary.

In the present specification “increased production” refers to increasedformation, increased release, or increased content of an endogenouscompound locally, regionally or systemically in a patient compared tothe content of said endogenous compound in a healthy individual. In thepresent specification “upregulated” refers to an increased activity orcontent of the compound compared with that in a healthy individual.

In the present specification “decreased production” is refers todecreased formation, decreased release, or decreased content of anendogenous compound in a patient compared to the content of saidendogenous compound in a healthy individual. In the presentspecification “downregulated” refers to a decreased activity or contentof the compound compared with that in healthy individual.

In particular, positive treatment effects or preventive effects are seenin conditions where inflammation or inflammatory like condition iscaused by or being associated with one or more of the following:allergy, hypersensitivity, bacterial infection, viral infection,inflammation caused by toxic agent, fever, autoimmune disease, radiationdamage by any source including UV-radiation, X-ray radiation,γ-radiation, α- or β-particles, sun burns, elevated temperature,mechanical injury. Moreover, inflammation due to hypoxia, which isoptionally followed by reoxygenation of the hypoxic area, is typicallyfollowed by severe inflammation, which condition is positively affectedby treatment with a compound of the invention.

In very specific embodiments of the invention a compound of theinvention is administered for prevention or therapeutic treatment ofinflammatory diseases of the skin (including the dermis and epidermis)of any origin, including skin diseases having a inflammatory component.Specific examples of this embodiment of the invention include treatmentof contact dermatitis of the skin, sunburns of the skin, burns of anycause, and inflammation of the skin caused by chemical agent, psoriasis,vasculitis, pyoderma gangrenosum, discoid lupus erythematosus, eczema,pustulosis palmo-plantaris, and phemphigus vulgaris.

Also comprised by the invention is the administration of the compound ofthe invention for treatment of an inflammatory disease in the abdomen,including an abdominal disease having a inflammatory component. Specificexamples of treatment of such disease with a compound of the inventionare gastritis, including one of unknown origin, gastritis perniciosa(atrophic gastritis), ulcerous colitis (colitis ulcerosa), morbus Crohn,systemic sclerosis, ulcus duodeni, coeliac disease, oesophagitis andulcus ventriculi.

Comprised by the invention is also administration of the compound of theinvention for treatment of systemic or general and/or localimmunological diseases, including those of an autoimmune nature, andother inflammatory diseases of a general nature. Specific examplesinclude treatment of rheumatoid arthritis, psoriatic arthritis, systemicsclerosis, polymyalgia rheumatica, Wegener's granulomatosis,sarcoidosis, eosinophilic fasceitis, reactive arthritis, Bechterew'sdisease, systemic lupus erythematosus, arteritis temporalis, Behcet'sdisease, morbus Burger, Good Pastures' syndrome, eosinophilic granuloma,fibromyalgia, myositis, and mixed connective tissue disease. Includedtherein is also arthritis, including arthritis of unknown origin.

Further included in the invention is administration of the compound ofthe invention for treatment of a disease of the peripheral and centralnervous system related to inflammation. Included in this aspect of theinvention is the treatment of cerebral vasculitis, multiple sclerosis,autoimmune ophtaimitis, polyneuropathia. Comprised by the invention isalso the administration of a compound of the invention for treatment ofan inflammation of the central nervous system to prevent apoptotic celldeath. Moreover, as the compounds of the invention show a distinctability to induce nerve regeneration, positive treatment effects areoften seen in central nervous system diseases involving damage of cellsin this region. This aspect of the invention also includes treatment oftraumatic injuries to the central nervous system, brain edema, multiplesclerosis, Alzheimer's disease, bacterial and viral infections in thecentral nervous system, stroke, and haemorrhagia in the central nervoussystem.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of diseases of the eye and tear glandsrelated to inflammation. Specific examples of such diseases compriseanterior and posterior uveitis, retinal vasculitis, otpicus neuritis,Wegener's granulomatosis, Sjögren's syndrome, episcleritis, scleritis,sarcoidosis affecting the eye and polychondritis affecting the eye.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of diseases of the ear related toinflammation, specific examples of which include polychondritisaffecting the ear and external otitis.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of diseases of the nose related toinflammation, specific examples of which are sarcoidosis, polychondritisand mid-line granuloma of the nose.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of diseases related to inflammation of themouth, pharynx and salivary glands. Specific examples include Wegener'sgranulomatosis, mid-line granuloma, Sjögren's syndrome andpolychondritis in these areas.

Included in the invention is also the administration of the compound ofthe invention for treatment of diseases related to inflammation in thelung. Specific examples include treatment of idiopathic alveolitis,primary pulmonary hypertension, bronchitis, chronic bronchitis,sarcoidosis, alveolitis in inflammatory systemic disease, pulmonaryhypertension in inflammatory systemic disease, Wegener's granulomatosisand Good Pastures' syndrome.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of diseases related to the inflammation ofthe heart. Specific examples include treatment of pericarditis,idiopathic pericarditis, myocarditis, Takayasus' arteritis, Kawasaki'sdisease, coronary artery vasculitis, pericarditis in inflammatorysystemic disease, myocarditis in inflammatory systemic disease,endocarditis and endocarditis in inflammatory systemic disease.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of diseases related to inflammation of theliver. Specific examples include treatment of hepatitis, chronic activehepatitis, biliary cirrhosis, hepatic damage by toxic agent, interferoninduced hepatitis, hepatitis induced by viral infection, liver damageinduced by anoxia, liver damage caused by mechanical trauma.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of diseases related to inflammation of thepancreas. Specific examples include treatment (and prevention) ofdiabetes-mellitus, acute pancreatitis, chronic pancreatitis.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of diseases related to the inflammation ofthe thyroidea. Specific examples of these embodiments of the inventioninclude treatment of thyreoiditis, autoimmune thyreoiditis andHashimoto's thyreoiditis.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of diseases related to inflammation of thekidney. Specific examples include treatment of glomerulonephritis,glomerulonephritis in systemic lupus erythematosus, periarteritisnodosa, Wegener's granulomatosis, Good-Pastures' syndrome, HLAb27associated diseases, IgA nephritis (IgA=Immunoglobuline A),pyelonephritis, chronic pyelonephritis and interstitial nephritis.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of diseases related to the inflammation ofthe joints. Specific examples include treatment of Bechterew's disease,psoriatic arthritis, rheumatoid arthritis, arthritis in colitisulcerosa, arthritis in morbus Crohn, affection of joints in systemiclupus erythematosus, systemic sclerosis, mixed connective tissuedisease, reactive arthritis, Reiter's syndrome. Moreover, included inthis embodiment of the invention is treatment of arthrosis of any joint,in particular arthrosis of finger joints, the knee and the hip.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of diseases related to the inflammation ofblood vessels. Specific examples include treatment of arteritistemporalis, periarteritis nodosa, arteriosclerosis, Takayasus' arteritisand Kawasaki's disease. In particular advantageous is the capacity of acompound of the invention to afford protection against and prevention ofarteriosclerosis. This is in part due to the capacity of the compound ofthe invention to prevent the induction of inducible nitric oxidesynthase (iNOS) caused by the action of oxidized Low Density Lipoproteinon endothelial cells and blood vessel walls.

Comprised by the invention is also the administration of a compound ofthe invention for treatment of drug induced disorders of the blood andlymphoid system, including the treatment of drug inducedhypersensitivity (including drug hypersensitivity) affecting blood cellsand blood cell forming organs (e.g. bone marrow and lymphoid tissue).Specific embodiments of this aspect of the invention include thetreatment of anemia, granulocytopenia, trombocytopenia, leukopenia,aplastic anemia, autoimmune hemolytic anemia, autoimmunethrombocytopenia and autoimmune granulocytopenia.

The compounds of the invention can also be administered for treatment offast allergic disorders (Type 1 allergy). Included in this embodiment ofthe invention is the treatment of anaphylactic reactions, anaphylactoidreactions, asthma, asthma of allergic type, asthma of unknown origin,rhinitis, hay fever and pollen allergy.

Comprised by the invention is also the administration of the compound ofthe invention for the treatment of inflammation related to infections ofany origin. Specific examples include treatment of inflammationsecondary to infection caused by virus, bacteria, helminths andprotozoae.

Comprised by the invention is also the administration of the compound ofthe invention for treatment of inflammations related to trauma andtissue injury of any origin.

Because of the capacity of the compounds of the invention to stimulatepigment formation in epidermal cells the compounds of the invention arealso useful for inducing skin tanning for cosmetic reasons, fortreatment of vitiligo, or any other condition where darkening of skincolor is desired. Moreover, because of the ability of the compounds ofthe invention to inhibit pigment formation in cells of the skin they arealso useful for inducing lighter skin color for cosmetic reasons, orduring any condition where a lighter color of skin is desired.

The compounds of the invention are also useful for inducing formation ofthe second messenger element cAMP. In particular, such formation ofcyclic adenosine 3′,5′-monophosphate (cAMP) is desired for eliciting thespecific pharmacological effects of the compounds of the invention whenadministered to a living organism, in particular a human. However, theinduction of cAMP formation may also be of great value in cells orcrushed cell systems in vitro, e.g. for analytical or diagnosticpurposes. A specific embodiment of this aspect of the invention is givenin Example 3.

The compounds of the invention are also useful for inhibiting the invivo formation of the second messenger element CAMP. Such inhibition mayalso be used in cells or crushed cell systems in vitro, e.g. foranalytical or diagnostic purposes.

The compounds of the invention are administered in pharmacologicallyeffective amounts which may vary from 0.001 mg/day/kg body weight to1000 mg/day/kg body weight depending on the nature of the compound, thedesired treatment effect and route of administration; however, loweramounts may be effective, if delivered locally. The compounds of theinvention have low toxicity and are well tolerated.

For analytical and diagnostic purposes the compounds of the inventioncan be used in radioactive form, including radioactive labels. Inparticular they may be manufactured so as to incorporate radioactiveiodine or tritium, or any other suitable radionuclide. Such aradioactively labeled compound can be used in radioligand binding forthe quantification of MC1-receptors, for the analysis of dissociationconstant (K_(i)s or K_(d)s) of drugs competing with specific subtypes ofmelanocortin receptors, and for the localization of MC1-receptors intissues and tissue sections e.g. by use of receptor autoradiographictechniques. Principles of radio-ligand binding and receptorautoradiography are well known in the art.

Alternatively the compounds of the invention can be labeled with anyother type of label that allows detection of the respective compound,e.g. a fluorescent label, biotin, an enzyme, and the resulting compoundbe used for the same purpose as the corresponding radioactively labeledcompound. The compound of the invention may also be labeled with otherlabels that are activated by secondary processes like irradiation withγ-radiation, light photons or by biochemical processes, the purposebeing to cause changes in a target cell or tissue wherein the labeledcompound of the invention is located. Such changes may be useful toaffect the viability and fate the cells or tissues in an organism towhich the compound is administered.

The compounds of the invention can also be provided with group that canbe activated by light, in particular UV-light, the purpose being toobtain a compound useful for covalent labeling of MC1-receptors by useof the photoaffinity labeling technique. Photoaffinity labeling is atechnique well known in the art which in the present context is usefulfor elucidating the structure and topological organisation of theMC1-receptor. Such photoactive derivatives of the compounds of theinvention are also part of the present invention. Photoactivederivatives of the compounds of the invention may also be made toincorporate and easily detectable group or label, such as e.g. aradioactive atom, a fluorescent group or biotin. (For further details inregard of photoaffinity labeling see Leeb-Lundberg et al. J. Biol. Hem.1984, 259, 2579 and Scimonelli & Eberle FEBS Lett 1978, 226, 134).

The compounds of the invention can also be labeled with gamma orposition emitting isotopes. A thus labeled compound may be administeredsystematically or locally to an animal, including a human. By virtue ofthe ability of the radioactive compound of the invention to bindselectively to MC1-receptors imaging of the in vivo binding of specificMC-receptor subtypes may be obtained by using techniques well known inthe art such as scintigraphy, positron emission tomography (PET) orsingle photon emission computed tomography (SPECT). By using suchmethods information on the distribution and/or numbers of MC1-receptorsin tissues of an animal or a human subject can be obtained. Thisinformation is of value for diagnosis of disease, in particular fordiagnosis of malignant melanoma, as melanoma cells are known to be richin MC1-receptors. Moreover, the very same technique may be used for thetreatment of any other malignancy where the malignant cells containMC1-receptors. Examples of the latter are cancer with originating fromthe ovaries or testes. For general examples of this technique, which isdirectly applicable for the compound of the invention, see Bagutt et al.1994.

Moreover, the compound of the invention can also be tagged with a toxicagent and used for targeted delivery of the toxin to malignant melanomacells or other cells bearing MC1-receptors (e.g. malignant cells withorigin from the ovary or testis). In this context ‘tagged’ is intendedto mean that the compound of the invention is covalently ornon-covalently bound to a toxin, for instance ricin, doxorubicin anddiphtheria toxin. By virtue of the high affinity and selectivity of thecompound of the invention for the MC1-receptor specific delivery of thetoxin to MC1-receptor bearing cells (e.g. melanoma cells) is achievedand malignant cell growth is selectively inhibited, even includingkilling of the malignant cells. The principles used for this approachare well known in the art (Murphy et al. 1986, Tatro et al. 1992, O'Hareet al. 1993). The compounds of the invention may also be tagged with acompound capable of activating the endogenous immune system. A thusformed hybrid of a compound of the invention and a immune systemactivating compound can be used for treatment of malignant melanoma orany other MC1-receptor expressing disease condition. The portion of thehybrid molecule containing the compound of the invention will, at theone hand, bind to the MC1-receptor of malignant melanoma cells(including both the primary tumor and metastases), as well as otherMC1-receptor bearing malignant tumor cells, while, at the other hand,the immune system activating component will trigger an immune responsedirected against the tumor cells. This turn leads to inhibition of tumorcell growth and eventual tumor cell death. Typical example of such a“bispecific” hybrid compound is a hybrid between the compound of theinvention [i.e. the compound according to formula (1), (2), (3) or (4)]and a compound capable of binding to a T-cell antigen. Typically theT-cell antigen binding compound of the hybrid compound is an antibodydirected against T-cell antigen CD3. The antibody in this case ispreferably a monoclonal antibody. The so formed hybrid compound willdirect cytotoxic cells to the malignant melanoma cells or theMC1-receptor bearing malignant cells and inhibit the tumor growth. Thecompound of the invention may be attached to the antibody chemically bycovalent or non-covalent bond(s). The hybrid compound may also bemanufactured by genetic engineering by incorporating the DNA sequenceencoding a peptide corresponding to the compound of the invention withother DNA encoding a suitable protein. The peptide sequence of thecompound of the invention may thus become incorporated into a longerpeptide sequence constituting, for instance, an antibody, a hybridantibody, a hybrid molecule containing part of an antibody, or anantibody like protein. It is furthermore recognized that the hybridcompound may contain one or several copies of the compound of theinvention. When several copies of the MC1-receptor binding moieties areincorporated into the hybrid compound of the invention the thus formedhybrid will show increased affinity to the MC1-receptor. This is anadvantage in the treatment of MC1-receptor related malignant disease. Inline with what was described above the present invention thereforeincludes a hybrid compound incorporating one or several copies of thecompound of the invention with another molecule. For the generalprinciples underlying immunotherapy using the approach described abovereference see Riedle et al. 1998, Mukherji et al. 1995, Canevari et al.1995 and Thielemans 1995. These approaches and methods can be adoptedfor targeting MC1-receptor expressing malignant conditions, by use ofthe approach outlined above.

The compounds of the invention may be used for the treatment anddiagnosis of diseases, disorders and/or pathological conditions in ananimal, in particular in man.

The compounds of the invention may be delivered to the preferred site inthe body by a suitable drug delivery system. For example a compound ofthe invention may be coupled to a carrier molecule to make it lipophilic(see e.g. Toth, I: Drug Targeting, 1994, 2, 217–239; Patel et al.,Bioconjugate Chem 1997, 8, 434–411). Other techniques useful fordelivering the compound of the invention to the desired site in the bodyare vector mediated carrier systems (see e.g. Pardridge, W M: PharmacolToxicol 1992, 71, 3–10; Saito, Y et al.: Proc. Natl. Acad. Sci USA 1995,92, 10227–10231; Wu, D and Pardridge, W M: J. Phammacol. Exp. Ther.1996, 279, 77–83). Yet other examples of drug delivery technologiesuseful for the compounds of the invention include the conjugation of thecompound of the invention with an active molecule capable of beingtransported through a biological barrier (see e.g. Zlokovic, B V:Pharmaceutical Research 1995, 12, 1395–1406). A specific exampleconstitutes the coupling of the compound of the invention to fragmentsof insulin in order to achieve transport across the blood brain barrier(Fukuta, M et al. Pharmaceutical Res. 1994, 11, 1681–1688). Otherexamples which are principally useful for and adaptable to the compoundsof the present invention are to be found in Prokai-Tatrai, K et al.: J.Med. Chem. 1996, 39, 4775–4782 and Tamai, I et al: J. Pharmacol. Exp.Ther. 1997, 280, 410415. For general reviews of technologies for drugdelivery suitable for the compounds of the invention see Zlokovic, B V:Pharmaceutical Res. 1995, 12: 1395–1406 and Pardrige, W M: Pharmacol.Toxicol. 1992, 71, 3–10.

The present invention also relates to a pro-drug which, uponadministration to an animal or a human, is converted to a compound ofthe invention. A pro-drug of the compound of the invention can be usedfor the same purposes as described in this patent for the compounds ofthe invention, as well as is disclosed in the Examples given below.

The compound of the invention can be administered together withpeptidase and/or protease inhibitors to prevent or delay the breakdownof the compound of the invention and thereby prolong its duration ofpharmacological action in the body as well as its stability in thegastrointestinal tract when administered perorally. Peptidase/proteaseinhibitors that may be administered together with a compound of theinvention are preferably selected from the group of angiotensinconverting enzyme inhibitors (ACE-inhibitors) such as e.g. captopril(D-3-mercaptomethyl-propionyl-L-proline), enaplapril, phosphoramidone,amastatin.

The compounds of the present invention can be bound covalently ornon-covalently to one or several of other molecule(s) of any desiredstructure(s); the thus formed modified compound or complex can be usedfor the same purposes as described in this patent for the compounds ofthe invention, as well as is disclosed in the Examples given below. In aparticularly important embodiment of the invention a radioactivelylabeled molecule is covalently bound to the compound of the invention soas to make the compound of the invention radioactively labeled.

In the following the invention will be described in greater detail byreference to a number of preferred embodiments which however are onlygiven for purposes of illustration and must not be considered to limitthe invention in any way.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Competion curves for MS05 and MS09 on MC1 and MC3 receptors.

FIG. 2: Inhibition of the binding of [¹²⁵I]-NDP-MSH to B16 melanomacells by MS05 and MS09.

FIG. 3: Stimulation of cAMP-formation in mouse B16 melanoma cells byα-MSH and MS05.

FIG. 4: Inhibition of the binding of [¹²⁵I]-NDP-MSH to mouse RAW 264.7cells by MS05.

FIG. 5: Stimulation of cAMP-formation in mouse RAW 264.7 cells by α-MSHand MS05.

FIG. 6: Inhibition of nitric oxide production in mouse RAW 264.7 cellsby α-MSH, MS05 and MS09.

ABBREVIATIONS

Abbreviations used in the following examples:

-   Fmoc=9-fluorenylmethoxycarbonyl-   DMF=N,N-Dimethylformamide-   Fmoc-Val-OPfp=9-Fluorenylmethoxycarbonyl-L-valine pentafluoro phenyl    ester-   HOAt=1-Hydroxy-7-azabenzotriazole-   Fmoc-Pro-OPfp=9-Fluorenylmethoxycarbonyl-L-proline pentafluorophenyl    ester-   Fmoc-Lys(Boc)-OPfp=α-9-Fluorenylmethoxycarbonyl-e-tert-butyloxycarbonyl-L-lysine    pentafluorophenyl ester-   Fmoc-Gly-OPfp=9-Fluorenylmethoxycarbonyl-glycine pentafluorophenyl    ester-   Fmoc-Trp(Boc)-OH=9-Fluorenylmethoxycarbonyl-(N^(in)-tert-butyloxycarbonyl)-L-tryptophan-   HATU=O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   DIEA=N,N-diisopropylethylamine-   Fmoc-Arg(Pbf)-OH=9-Fluorenylmethoxycarbonyl-(N^(g)-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine-   Fmoc-Phe-OPfp=9-Fluorenylm thoxycarbonyl-L-phenylalanine    pentafluorophenyl ester-   Fmoc-His(Trt)-OH=9-Fluorenylmethoxycarbonyl-N^(im)-trityl-L-histidine-   Fmoc-Ser(But)-OH=9-Fluorenylmethoxycarbonyl-O-tert-butyl-L-serine-   Fmoc-Ile-OPfp=9-Fluorenylmethoxycarbonyl-L-isoleucine    pentafluorophenyl ester-   Ac=acetyl-   dPhe=D-phenylalanine-   dSer=D-serine-   NMeSer=N-methyl-L-serine-   NMeVal=N-methyl-L-valine-   NMedPhe=N-methyl-D-phenylalanine-   PyAOP=O-(7-azabenzotriazol-1-yl)-tris(pyrrolidino)-phosphonium    hexafluorophosphate-   TFFH=tetramethylfluoroformamidinium hexafluorophosphate-   NDP-MSH=[Nle⁴,D-Phe⁷]α-MSH

EXAMPLE 1:1 Synthesis ofL-seryl-L-seryl-L-Isoleucyl-L-isoleucyl-L-seryl-L-histidyl-L-phenytalanyl-L-arginyl-L-tryptophanyl-glycl-L-lysyl-L-prolyl-L-valinamide(MS05) (SEQ ID NO:1):

The MS05 peptide(Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) (SEQ ID NO:1)was assembled on a solid support using the Pioneer peptide synthesissystem from PerSeptive Biosystems UK.

The starting cycle was as follows: 100 mg (0.02 mmole) of[5-(4-Fmoc-aminomethyl-3,5-dimethoxy) valeric acid attached topolyethylene-graft polystyrene support (Fmoc-PAL-PEG-PS, capacity 0.2mmole/g) was placed into a peptide synthesis column. The Fmoc group wasthen removed by 5 min treatment with 20% piperidine in DMF, followed bya wash of the support with DMF.

After completion of the starting cycle the resin was subjected torepeated aminoacid coupling cycles, each cycle consisting of 30–90 mincirculation of appropriate reagents (as detailed below) dissolved in 4ml DMF through the column, followed by washing with DMF, 5 min treatmentwith 0.3 M acetic anhydride in DMF, washing with DMF, 5 min treatmentwith 20% piperidine in DMF, and then again washing with DMF before thestart of the next cycle. Thirteen cycles were applied using reagents andtreatment times as follows (in order): 1) Fmoc-Val-OPfp (40 mg, 0.08mmol) and HOAt (11 mg, 0.08 mmol) (90 min), 2) Fmoc-Pro-OPfp (40 mg,0.08 mmol) and HOAt (11 mg, 0.08 mmol)(60 min), 3) Fmoc-Lys(Boc)-OPfp(51 mg, 0.08 mmol) and HOAt (11 mg, 0.08 mmol) (30 min), 4)Fmoc-Gly-OPfp (37 mg, 0.08 mmol) and HOAt (11 mg, 0.08 mmol)(30 min), 5)Fmoc-Trp(Boc)-OH (42 mg, 0.08 mmol), HATU (30 mg, 0.08 mmol) and DIEA(0.068 ml, 0.4 mmol) (30 min), 6) Fmoc-Arg(Pbf)-OH (52 mg, 0.08 mmol),HATU-(30 mg, 0.08 mmol) and DIEA (0.068 ml, 0.4 mmol) (90 min), 7)Fmoc-Phe-OPfp (44 mg, 0.08 mmol) and HOAt (11 mg, 0.08 mmol)(60 min), 8)Fmoc-His(Trt)-OH (50 mg, 0.08 mmol), HATU (30.0 mg, 0.08 mmol) and DIEA(0.068 ml, 0.4 mmol) (30 min), 9) Fmoc-Ser(tBu)-OH (31 mg, 0.08 mmol),HATU (30.0 mg, 0.08 mmol) and DIEA (0.068 ml, 0.4 mmol) (30 min), 10)Fmoc-Ile-OPfp (42 mg, 0.08 mmol) and HOAt (11 mg, 0.08 mmol) (90 min),11) Fmoc-Ile-OPfp (42 mg, 0.08 mmol) and HOAt (11 mg, 0.08 mmol) (90min), 12) Fmoc-Ser(tBu)-OH (31 mg, 0.08 mmol), HATU (30.0 mg, 0.08 mmol)and DIEA (0.068 ml, 0.4 mmol) (60 min), 13) Fmoc-Ser(tBu)-OH (31 mg,0.08 mmol), HATU (30.0 mg, 0.08 mmol) and DIEA(0.068 ml, 0.4 mmol) (30min).

After the last cycle the support was washed with DMF, followed by amethanol and methylene chloride wash, and dried in vacuo. The driedresin was treated with 2.5 ml of deprotection mixture (trifluoroaceticacid-phenol-anisole-1,2-ethanedithiol-water, 82:2:2:2:2) for 3 hours atroom temperature. It was filtered, washed on the filter withtrifluoroacetic acid, the filtrates combined and concentrated in vacuoat room temperature. Dry ether was added and the precipitate formed wasfiltered off and washed on the filter with ether, then dried in vacuoover KOH. Yield 31 mg. HPLC data (2×250 mm column, Vydac RP C18, 90A,201HS1010): k′(main substance)=3.50 (19% acetonitrile in water+0.1%trifluoroacetic acid, detection at 220 nm). The raw product wasdissolved in 1 ml of 60% MeCN in water and the solution divided intothree portions and placed into centrifuge tubes, each of them then beingdiluted with 0.1% aqueous trifluoroacetic add to 1.5 ml volume. It wascentrifuged and the clear solutions were used for semipreparative HPLC(10×250 mm column, Vydac RP C18, 90A, 201HS1010, eluate −19%acetonitrile in water +0.1% trifluoroacetic acid, detection at 220 nm.Fractions containing the main peak were pooled and lyophilized. A whitepowder was formed. Yield of trifluoroacetate salt of MS-05 was 13.2 mg(33%). R_(f) 0.28(1-butanol-pyridine-acetic acid-water, 4:1:1:2). Massspectrometry data: m/e=1514.3.

EXAMPLE 1:2

Synthesis of Ser-Ser-Ile-Ile-Ser-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-09) (SEQ ID NO:2) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Yield 36%. R_(f) 0.56. k′ 2.0 (21% MeCN in 0.1% TFA). m/e 1512.9.

EXAMPLE 1:3

Synthesis of Tyr-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-30) (SEQ ID NO:3) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Yield 33%. R_(f) 0.79. k′ 4.4 (8.4% MeCN in 0.1% TFA). m/e 1675.9.

EXAMPLE 1:4

Synthesis of Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val NH₂(MS-31) (SEQ ID NO:4) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Yield 34%. R_(f) 0.75. k′ 2.7 (10.8% MeCN in 0.1% TFA). m/e 1589.1.

EXAMPLE 1:5

Synthesis of Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Tyr-NH₂(MS-32) (SEQ ID NO:5) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Yield 28%. R_(f) 0.75. k′ 3.3 (9.6% MeCN in 0.1% TFA). m/e 1675.9.

EXAMPLE 1:6

Synthesis of Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro Val-NH₂ (MS-33)(SEQ ID NO:6) tetratrifluoroacetate was made essentially as described inExample 1.

Yield. 26%. R_(f) 0.73. k′ 3.6 (8.4% MeCN in 0.1% TFA). m/e 1425.7.

EXAMPLE 1:7

Synthesis of Thr-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-34) (SEQ ID NO:7) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Yield 52%. R_(f) 0.72. k′ 2.4 (10.8% MeCN in 0.1% TFA). m/e 1527.1.

EXAMPLE 1:8

Synthesis of Ser-Thr-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-35) (SEQ ID NO:8) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Yield 49%. R_(f) 0.68. k′ 2.2(10.8% MeCN in 0.1% TFA). m/e 1526.8.

EXAMPLE 1:9

Synthesis of Ser-Ser-Val-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-36) (SEQ ID NO:9) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Yield 42%. R_(f) 0.68. k′ 2.3 (10.8% MeCN in 0.1% TFA). m/e 1499.3.

EXAMPLE 1:10

Synthesis of Ser-Ser-Ile-Val-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-37) (SEQ ID NO:10) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Yield 69%. R_(f) 0.65. k′ 3.3 (12.0% MeCN in 0.1% TFA). m/e 1499.0.

EXAMPLE 1:11

Synthesis of Ac-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-38) (SEQ ID NO: 11) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Yield 49%. R_(f) 0.72. k′ 1.8 (10.8% MeCN in 0.1% TFA). m/e 1554.7.

EXAMPLE 1:12

Synthesis of dSer-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-39) (SEQ ID NO:12) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Yield 44%. R_(f) 0.69. k′ 2.2 (12% MeCN in 0.1% TFA). m/e 1512.4.

EXAMPLE 1:13

Synthesis of NMeSer-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-40) (SEQ ID NO:13) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Fmoc-NMeSer(Bu^(t))-OH was added using PyAOP.

Yield 47%. R_(f) 67. k′ 1.7 (12% MeCN in 0.1% TFA). m/e 1526.6.

EXAMPLE 1:14

Synthesis of Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-NMeVal-NH₂(MS-41) (SEQ ID NO:14) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Fmoc-NMeVal-OH was added using TFFH.

Yield 2.5%. R_(f) 0.68. k′ 4.4 (22.8% MeCN in 0.1% TFA). m/e 1527.3.

EXAMPLE 1:15

Synthesis of Ser-Ser-Ile-Ile-Ser-His-NMedPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-42) (SEQ ID NO:15) tetratrifluoroacetate was made essentially asdescribed in Example 1.

Fmoc-NMedPhe-OH was added using PyAOP.

Yield 40%. R_(f) 0.70. k′ 2.5 (10.8% MeCN in 0.1% TFA). m/e 1527.0.

EXAMPLE 2

Assay of Binding Affinities of Compounds of the Invention for HumanMC-Receptors

Expression of receptor clones. Human MC1- and MC5-receptor DNAs(Chhajlani and Wikberg 1992; Chhajlani-et al., 1993), cloned into theexpression vector pRc/CMV (InVitrogen Corp., USA), and human MC3- andhuman MC4-receptor DNAs (Gantz et al., 1993a & b), cloned into theexpression vector pCMV/neo, were used. COS cells were grown andtransfected with receptor clones as described (Schiöth et al. 1995,1996b). After transfection cells were cultivated for 48 h, detached fromthe Petri dishes, and used for radioligand binding as described (Schiöthet al. 1995, 1996b).

Binding studies. The transfected cells were washed with binding buffer(Minimum Essential Medium with Earle's salts, 25 mM HEPES, pH 7.0, 0.2%bovine serum albumin and distributed into 96 well plates. The cells werethen incubated for 2 h at 37° C., with 0.1 ml binding buffer in eachwell containing [¹²⁵I][Nle⁴, D-Phe⁷]α-MSH and appropriate concentrationsof the peptide to be tested. After incubation the plates were put on iceand the cells were washed with 0.1 ml of ice-cold binding buffer. Thecells were then detached from the plates with 0.2 ml of 0.1 N NaOH.Radioactivity was counted by using a Wallac, Wizard automatic gammacounter. The competition data were analyzed by fitting it to thelogistic function using non-linear regression analysis. The K_(i)-valueswere then calculated from the thus obtained IC50-values by using theCheng and Prusoff equation, essentially as described by Schiöth et al.1995, 1996b.

Results

K_(i) values for NDP-MSH, α-MSH, MS05 and MS09 for MC1, MC3, MC4 and MC5receptors are shown in the Table below:

MC1 MC3 MC4 MC5 Compound K_(i) (nM) K_(i) (nM) K_(i) (nM) K_(i) (nM)NDP-MSH 0.11 0.39 2.3 2.9 α-MSH 0.68 52.3 2030 4990 MS05 0.761365 >>50000 >>50000 MS09 0.20 7.0 40 120

FIG. 1 shows competition curves for MS05 and MS09 on MC1 and MC3receptors.

EXAMPLE 3

Assays of the Effect of the Compounds of the Invention on Generation ofthe Second Messenger cAMP in Cells Expressing MC1-Receptors

Preparation of Stable Cell Lines

Human MC1-receptors had been cloned earlier into pZeoSV (Invitrogen)(Szardenings et al., 1997). The resulting vectors were used fortransformation of the amelanotic mouse melanoma cell line B16G4F thatlacks the mouse MC1-receptors (Solca et al. 1993) using liposomes(Campbell, 1995) as described previously (Schiöth et al., 1997b). Cellswere cultivated in Dulbecco's modified Eagle's medium with 10% fetalcalf serum at 7% CO₂ for 24 hours and seeded on large plates in the samemedium containing 300 μg/ml Zeocin. The medium was exchanged every 4–5days until cell foci appeared. Single foci were isolated and grown underantibiotic selection. Expression and binding characteristics of thesereceptors were determined and found to be identical with the samereceptors expressed in other cell lines, as has already also been shownearlier for the human MC1-receptor (Chluba-de-Tapia et al. 1996).

Stimulation of Cell cAMP

For CAMP measurements the cells were detached from 60–80% confluentadherent cultures using Hank's balanced salts containing 0.5 mM EDTA andincubated for 30–60 min at 37° C. in ordinary growth medium containing0.5 mM of the phosphodiesterase inhibitor 3-iso-butyl-1-methyl-xanthine(IBMX). 20 μl aliquots of appropriate dilutions of test compounds ingrowth medium were prepared in 96 well microtitre plates and placed in awater bath at 37° C. For the stimulation about 1.5×10⁵ cells in 180 μlwere quickly added to each well to obtain immediate mixing. After 20 min20 μl of 4.4 M perchloric acid were added, mixed, neutralized after afew minutes by addition of 20 μl base (5 M KOH, 1 M Tris) andcentrifuged.

Determination of cAMP Concentrations

20 μl of acid treated supernatant obtained above were mixed with 50 μlbuffer (100 mM Tris-Cl, 250 mM NaCl, 10 mM EDTA, 0.1% mercaptoethanol,0.5 mM IBMX, pH=7.4) containing 0.01 μCi [³H]CAMP (Amersham, 1.04TBq/mmol, 1 μCi/μl, product no.: TRK304). 200 μl of the same buffercontaining a 1:16 diluted porcine adrenal gland bark extract (preparedas described by Nordstedt and Fredholm, 1990) were added and themicrotitre plates were incubated for at least 2 hours at 4° C. Astandard curve was prepared in the same manner with dilutions of CAMPcovering the range 2 μM –0.5 nM.

After completion of incubation the solutions were filtered over GF-Bglassfibre filters (Whatman) and washed briefly with ca. 2 ml ice-coldwashing buffer (50 mM Tris-Cl, pH=7.4). Radioactivity on the filters wasmeasured after addition of scintillation liquid. Stimulation experimentswere determined in quadruplicates and standard curves in duplicates.

Results of Tests of MS05 and MS09 on cAMP Formation in the MC1-ReceptorExpressing Cells

The magnitude of stimulation of cAMP formation in the treatedMC1-receptor expressing cells compared to the CAMP content in untreatedcontrol cells were tested for 10 nM and 1 μM MS05 and MS09, and were asgiven in the below table. For comparison the stimulation obtained byNDP-MSH, α-MSH, forskolin and MS04 (Szardenings et al. 1997) is alsogiven.

Fold stimulation % of 10 nM Compound of cAMP α-MSH NPD-MSH (10 nM) 7.4 ±0.7 123%  NPD-MSH  (1 μM) 5.9 ± 0.4 α-MSH (10 nM) 6.2 ± 0.7 100%  α-MSH (1 μM) 5.0 ± 0.6 MS04 (10 nM) 1.0 ± 0.1  0% MS04  (1 μM) 4.6 ± 0.4 MS05(10 nM) 4.6 ± 0.3 69% MS05  (1 μM) 6.7 ± 0.3 MS09 (10 nM) 5.5 ± 0.9 89%MS09  (1 μM) 7.0 ± 0.4 Forskolin  (1 μM) 7.3 ± 1.1 Control 1

In the above table the foldness of stimulation (compared to untreatedcontrol cells) is shown as the mean±standard deviation of 3 independentexperiments. Shown is also the ability of 10 nM of a compound tostimulate CAMP calculated in percent (%) of the capacity of 10 nM ofα-MSH to induce stimulation of CAMP. It is evident from the above tablethat both MS05 and MS09 are effective stimulators of CAMP formation atboth 10 nM and 1 μM concentration. Note that the MS04 peptide iscompletely devoid of effect on CAMP at 10 nM and 1 μM of MS04 isrequired to induce stimulation of CAMP formation. Both MS05 and MS09 arehighly effective stimulators of cAMP at 10 nM.

EXAMPLE 4

Demonstration of the Capacity of the Compounds of the Invention to Bindto Melanocortin (MSH) Receptors in Mouse B16 Melanoma Cells

Cell Culture

B16 mouse melanoma cells (B16-F1; CR-6323, American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., 20852) were culturedin Dulbecco's modified Eagle medium (DMEM; Gibco BRL, Gaithersburg, USA,cat no. 041-01966H) supplemented with 10% heat inactivated fetal bovineserum, 100 IU penicillin/ml and 100 microgram streptomycin/ml at 37° C.in a humidified atmosphere of 95% air and 5% CO₂. Cells grown inmonolayers were detached from the culture flasks using Hank's balancedsalts containing 0.5 mM EDTA and collected by low speed centrifugation(700×g).

Receptor Binding Studies

Assays for MSH-receptor binding was done essentially as described (Xiaet al., Cancer Letters, 1996, 98, 157–162), in principle according toearlier described methods (Eberle et al., J. Recept. Res. 1991, 11,311–322). In brief, collected cells were washed with binding buffer (forcomposition of binding buffer see below) distributed into 96 well platesand sedimented onto the well bottoms by centrifugation. The cells werethen incubated for 2 h at 37° C., with 0.1 ml binding buffer in eachwell, the buffer containing [¹²⁵I][Nle⁴, D-Phe⁷] α-MSH (0.2 nM),different concentrations of the test compound in different wells at 37°C. in binding buffer of the following composition MEM (MEM=MinimumEssential Medium, Gibco BRL, Gaithersburg, USA, cat no. 041-01095H) withEagle's salts, 25 mM Hepes, pH 7.4, 0.2% bovine serum albumin, 1 mM1,10-phenanthroline, 0.5 microgram leupeptin/ml and 200 microgrambacitracin/ml. After incubation the plates were put on ice, centrifugedand the cells washed with 0.1 ml of ice-cold binding buffer, centrifugedand the binding buffer was sucked off. The finally sedimented and washedcells were then detached from the plates with 0.2 ml of 0.1 N NaOH.Radioactivity was counted by using a Wallac, Wizard automatic gammacounter. The competition data were analysed by law of massactioncomputer modelling essentially as described (Bergstrom & Wikberg, ActaPharmacol. Toxicol. 1986, 59, 270–278).

Results

As is seen from FIG. 2 both the MSO5 and the MSO9 peptide caused a dosedependent inhibition of the binding of [¹²⁵]-NDP-MSH to the B16 melanomacells. Analysis of data by law of massaction computer modellingindicated that the MSO5 and MSO9 peptides competed with ¹²⁵I]-NDP-MSH atone single class of binding site (i.e. the B16 melanoma cellMSH-receptor; in other words the native mouse MC1-receptor). The K_(i)of MS05 for the mouse B16 melanoma cell MSH-receptor was estimated to be1.00±0.11 nM (mean±SEM; n=2). The K_(i) of MSO9 for the mouse B16melanoma cell MSH receptor was estimated to be 0.060±0.0035 nM(mean±SEM; n=2).

EXAMPLE 5

Demonstration of the Capacity of the Compounds of the Invention toAfford Stimulation of cAMP-Formation in Mouse B16 Melanoma Cells

Cell Culture

B16 mouse melanoma cells (B16-F1; CRL-6323, American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., 20852), were culturedin Dulbecco's modified Eagle medium (DMEM; Gibco BRL, Gaithersburg, USA,cat no. 041-01966H) supplemented with 10% heat-inactivated fetal bovineserum, 100 IU penicillin/mL and 100 μg streptomycin/mL at 37° C. in ahumidified atmosphere of 95% air and 5% CO₂. Cells grown in monolayerswere detached from 60–80% confluent adherent cultures using Hank'sbalanced salts containing 0.5 mM EDTA and collected by low speedcentrifugation (700×g).

Stimulation of Cell cAMP

The cells were incubated for 30 min at 37° C. in DMEM containing 0.5 mMof the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX).20 μL aliquots of appropriate dilutions of test compounds in growthmedium were prepared in 96 well microtitre plates. The plates were thenplaced in a water bath at 37° C. and about 1.5×10⁵ cells in 180 μL werequickly added to each well to obtain immediate mixing. After 20 min, 20μL 4.4 M perchloric acid was added to each well with mixing, and after afew minutes 20 μL base (5 M KOH, 1 M Tris) was added to each well,whereafter the plates were centrifuged, and the supernatants collected.

Determination of cAMP Concentrations

Twenty μL of the above supernatants were mixed with 50 μL cAMP assaybuffer (100 mM Tris-Cl, 250 mM NaCl, 10 mM EDTA, 0.1% mercaptoethanol,0.5 mM IBMX, pH 7.4) containing 0.01 μCi [³H] CAMP (Amersham, 1.04TBq/mmol, 1 μCi/μL, product no.: TRK3O4) in 96 microtiter plate wells.200 μL of cAMP assay buffer containing a 1:16 diluted porcine adrenalgland bark extract (prepared as described by Nordstedt and Fredholm,Anal. Chem, 1990, 189, 231–234) was then added into each well, and themicrotitre plates were incubated for at least 2 hours at 4° C. Astandard curve was prepared by exchanging the supernatants with CAMPstandards covering the concentration range 2 μM –0.5 nM of final CAMP inthe assays.

After completion of the incubations the solutions were rapidly filteredonto GF-B glassfibre filters (Whatman), followed by rapid washing offilters with 2 ml ice-cold washing buffer (50 mM Tris-Cl, pH 7.4).Filters were then placed into scintillation vials and a scintillationcocktail was added. Radioactivity was counted using a β-counter. Cellexperiments were determined in quadruplicates and standard curves induplicates.

Results

The results from the test of α-MSH and MSO5 peptide on CAMP levels inmouse B16 melanoma cells is shown in FIG. 3. As can be seen from thefigure, both peptides caused a marked increase in CAMP at similarpotencies and efficacies.

EXAMPLE 6

Demonstration of the Capacity of the Compounds of the Invention to Bindto Melanocortin (MSH) Receptors in RAW 264.7 Macrophage Cells.

Cell Culture

RAW 264.7 cells (TIB-71) were obtained from American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., 20852, USA, andcultured in Dulbecco's modified Eagle medium (DMEM; Gibco BRL,Gaithersburg, USA, cat no. 041-01966H) supplemented with 10%heat-inactivated fetal bovine serum, 100 IU penicillin/ml and 100 μgstreptomycin/ml at 37° C. in a humidified atmosphere of 95% air and 5%CO₂. Cells grown in monolayers were detached from the culture flasks andcollected by low speed centrifugation (700×g).

Receptor Binding Studies

MSH-receptor binding was done essentially as described (Xia et al.,Cancer Letters, 1996, 98, 157–162), in principle according to earlierdescribed methods (Eberle et al., J. Recept.

Res. 1991, 11, 311–322). In brief, the collected cells were washed,distributed into 96 well plates and sedimented onto the well bottoms bycentrifugation. The cells were then incubated for 2 h at 37° C., with0.1 ml binding buffer in each well containing [¹²⁵I][Nle⁴, D-Phe⁷]α-MSH(0.1 nM), different concentrations of the test compound in differentwells at 37° C. in MEM (Minimum Essential Medium) with Eagle's salts, 25mM Hepes, pH 7.4, 0.2% bovine serum albumin, 1 mM 1,10-phenanthroline,0.5 microgram leupeptin/ml and 200 microgram bacitracin/ml. Afterincubation the plates were put on ice, centrifuged and the cells washedwith 0.1 ml of ice-cold binding buffer, centrifuged and the bindingbuffer was sucked off. The finally sedimented and washed cells were thendetached from the plates with 0.2 ml of 0.1 N NaOH. Radioactivity wascounted by using a Wallac, Wizard automatic gamma counter. Thecompetition data were analysed by law of massaction computer modellingessentially as described (Bergstrom & Wikberg, Acta Pharmacol. Toxicol.1986, 59, 270–278).

Results

As is seen from FIG. 4, the MSO5 peptide caused a dose dependentinhibition of the binding of [¹²⁵I]-NDP-MSH to the mouse RAW 264.7cells. Analysis of data by law of massaction computer modellingindicated that MSO5 competed with [¹²⁵I]-NDP-MSH at one single class ofbinding site (i.e. the RAW 264.7 cell MSH-receptor, which in other wordsis the native mouse MC1 receptor of the RAW 264.7 cells). Thedissociation constant (K_(i)) of MS05 for the binding site was estimatedto be 1.35±0.61 nM (mean±SEM).

EXAMPLE 7

Demonstration of the Capacity of the Compounds of the Invention toAfford Stimulation of cAMP-Formation in Mouse RAW 264.7 Macrophage Cells

RAW 264.7 cells were grown as described in Example 6. CAMP wasdetermined essentially as described in Example 5. Results for α-MSH andMSO5 peptide are shown in FIG. 5. As can be seen from the Figure, bothpeptides caused a marked increase in cAMP at similar potencies andefficacies.

EXAMPLE 8

Demonstration of the Capacity of the Compounds of the Invention toInhibit Nitric Oxide Production in Mouse RAW 264.7 Macrophage Cells.

Cell Culture

RAW 264.7 cells (TIB-71), obtained from American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., 20852, USA, andcultured in Dulbecco's modified Eagle medium (Gibco, BRL) supplementedwith 10% heat-inactivated fetal bovine serum, 100 IU penicillin/ml and100 μg streptomycin/ml at 37° C. in a humidified atmosphere of 95% airand 5% CO₂. Cells grown in monolayers were detached from the cultureflasks and collected by low speed centrifugation (700×g).

Incubation of Compounds of the Invention with RAW 264.7 Cells

The cells obtained as above were resuspended in F-12 (HAM) medium(Gibco, BRL) and distributed into 96-well plates at a density of 2.5×10⁶cells per well, and incubated with 100 ng/mL bacteriallipopolysaccharide (L4391, Sigma Chemical Company, P.O. Box 14508, St.Louis, Mo. 63178, USA), 5 units/mL of mouse recombinant interferon gamma(15517, Sigma Chemical Company, P.O. Box 14508, St Louis, Mo. 63178,USA) and the compounds of the invention using concentrations ranging 01μM, for 16 h, whereafter an aliquot of the medium was collected formeasurement of nitric oxide (NO).

Measurement of Nitric Oxide

Nitric oxide was measured by monitoring the nitrite productionessentially using the method of Wishnok et al. (Methods in Enzymology,1996, 268, 130–151). In brief 50 μL of culture medium was mixed with 50μL Griess reagent (i.e. a 1:1 mixture of 0.1% N-naphthylethylenediaminedihydrochloride and 1% sulfanilamide in 5% (v/v) phosphoric acid) andafter 10 min the absorption was measured at 540 nm. The nitriteconcentrations were calculated from a standard curve constructed, byinstead of culture medium, adding 50 μL of between 3 to 100 μM of NaNO₂to the assays.

Results

The results are shown in FIG. 6. As can be seen from the Figure, thecompounds of the invention, MSO5 and MSO9, as well as α-MSH caused astrong dose dependent inhibition of the NO-production, the potencies andefficacies of MSO5 and MSO9 being similar to that of the α-MSH. Thisdata shows that MSO5 and MSO9 share the capacity of α-MSH to inhibitinflammation. This is because NO is a key component of inflammation.

1. A compound of general formula (1):

wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 areselected independently from H and methyl, and wherein R13, R14, R15 andR16 are selected independently from H and alkyl and wherein optionallyone hydrogen in R13 and one hydrogen in R14 is exchanged for a bondbetween R13 and R14, and wherein optionally one hydrogen in R15 and onehydrogen in R16 is exchanged for a bond between R15 and R16, and whereinL1 and L2 are linkers which are independently selected from the groupconsisting of single bond, methyl, and ethyl, and wherein R19, R20 andR21 are selected independently from H and —CH₂X, where X is H, alkyl,substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl,substituted alkenyl, heteroalkenyl, substituted heteroalkenyl, alkynyl,substituted alkynyl, heteroalkynyl, substituted heteroalkynyl,cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitutecycloheteroalkyl, cycloalkenyl, substitute cycloalkenyl,cycloheteroalkenyl, substituted cycloheteroalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, and functional group Q, whereQ is selected from the group consisting of amino, alkylamino,dialkylamino, arylamino, arylazido, heteroarylamino, heteroarylazido,hydroxy, alkylhydroxy, fluorinated alkylhydroxy, cyano, carboxy,alkylcarboxy, arylcarboxy, halogen, nitro, hydroxylamino, acyl,fluorinated acyl, nitroso, sulfonyl, sulfinyl, thio, alkylthio, andarylthio, and wherein NT is selected from H, hydroxyl, alkyl, aminoacid,aminoacid analogue, polypeptide and functional group Q, and CT isselected from hydrogen, hydroxyl, alkyl, aminoacid, aminoacid analogue,polypeptide and functional group Q, and wherein each asymmetric center(*) is in R or S configuration.
 2. The compound of claim 1, wherein R20is —CH₂X, wherein X is phenyl.
 3. The compound of claim 1, wherein oneor several of the nitrogens of the peptide backbone have been exchangedfor carbon substituted with hydrogen, and/or wherein one or several ofthe oxygens of the carbonyl groups of the peptide backbone has beenexchanged for two hydrogens.
 4. The compound of claim 1, having thestereomeric conformation given in the general formula (2):

.
 5. A compound according to claim 1, of formula (2) (SEQ ID NO:1):

.
 6. A compound, of the general formula (4):

wherein moiety A is optionally exchanged for hydrogen, hydroxyl, alkyl,aminoacid, aminoacid analogue, polypeptide, or functional group, whereinmoiety B is optionally exchanged for hydrogen, hydroxyl, alkyl,aminoacid, aminoacid analogue, polypeptide, or functional group, whereinoptionally moiety D is exchanged for aminoacid or aminoacid analogue,wherein optionally moiety E is exchanged for aminoacid or aminoacidanalogue, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12are selected independently from H and methyl, and wherein R13, R14, R15and R16 are selected independently from H and alkyl and whereinoptionally one hydrogen in R13 and one hydrogen in R14 is exchanged fora bond between R13 and R14, and wherein optionally one hydrogen in R15and one hydrogen in R16 is exchanged for a bond between R15 and R16, andwherein L1 and L2 are linkers which are independently selected from thegroup consisting of single bond, methyl, and ethyl, and wherein R19, R20and R21 are selected independently from H and —CH₂X, where X is H,alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl,substituted alkenyl, heteroalkenyl, substituted heteroalkenyl, alkynyl,substituted alkynyl, heteroalkynyl, substituted heteroalkynyl,cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitutecycloheteroalkyl, cycloalkenyl, substitute cycloalkenyl,cycloheteroalkenyl, substituted cycloheteroalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, and functional group Q, whereQ is selected from the group consisting of amino, alkylamino,dialkylamino, arylamino, arylazido, heteroarylamino, heteroarylazido,hydroxy, alkylhydrxy, fluorinated alkylhydroxy, cyano, carboxy,alkylcarboxy, arylcarboxy, halogen, nitro, hydroxylamino, acyl,fluorinated acyl, nitroso, sulfonyl, sulfinyl, thio, alkylthio, andarylthio, and wherein NT is selected from H, hydroxyl, alkyl, aminoacid,aminoacid analogue, polypeptide and functional group Q, and CT isselected from hydrogen, hydroxyl, alkyl, aminoacid, aminoacid analogue,polypeptide and functional group Q, and wherein each asymmetric center(*) is in R or S configuration.
 7. A compound according to claim 1,wherein one or several of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11and R12 are selected to be methyl, whereas the rest is selected to behydrogen, the selections being made so as to prevent or deceleratebreakdown by proteases and/or peptidases.
 8. A compound according toclaim 1, wherein less than 6 of the R1, R2, R3, R4, R5, R6, R7, R8, R9,R10, R11 and R12 are methyl.
 9. A compound comprising the sequenceSer-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS-05)(SEQ IDNO:1), wherein the amino-acids are all L-aminoacids; or a compoundcomprising the sequence:Ser-Ser-Ile-Ile-Ser-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS-09) (SEQ IDNO:2).
 10. A compound comprising one of the following sequences:Ser-Ser-Ile-Ile-Ser-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS-09) (SEQ IDNO:2), Tyr-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-30) (SEQ ID NO:3),Tyr-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS-31) (SEQ IDNO:4), Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Tyr-NH₂(MS-32) (SEQ ID NO:5),Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS-33) (SEQ IDNO:6), Thr-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS-34)(SEQ ID NO:7), Ser-Thr-Ile-Ile-Ser-His-Phe-Arg-Trp-GIy-Lys-Pro-Val-NH₂(MS-35) (SEQ ID NO:8),Ser-Ser-Val-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS-36) (SEQ IDNO:9), Ser-Ser-Ile-Val-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS-37)(SEQ ID NO:10),Ac-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS-38) (SEQID NO:11), dSer-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂(MS-39) (SEQ ID NO:12),NMeSer-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS40) (SEQID NO:13), Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-NMeVal-NH₂(MS41) (SEQ ID NO:14) orSer-Ser-Ile-Ile-Ser-His-NMedPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS42) (SEQID NO:15).
 11. A compound according to claim 1, R20 is —CH₂X, wherein Xis phenyl or substituted phenyl, wherein the compound is capable ofactivating MC1-receptors in vitro.
 12. A compound according to claim 1,R20 is —CH₂X, wherein X is naphthalene, or substituted naphthalene,wherein the compound is capable of blocking MC1-receptors in vitro. 13.A compound according to claim 9, which inhibits NO (nitric oxide)production, or the formation of nitrate.
 14. A compound according toclaim 9, which is immunomodulatory.
 15. A compound according to claim 9,which ameliorates or inhibits contact hypersensitivity.
 16. A compoundaccording to claim 9, which inhibits sensitization by a hapten.
 17. Acompound according to claim 9, which has the ability to induce haptentolerance.
 18. A compound according to claim 9, which ameliorates orinhibits formation of oedema.
 19. A compound according to claim 9, whichameliorates or inhibits inflammation of blood vessels or vasculitis. 20.A compound according to claim 9, which normalizes blood cell counts,said blood cell counts prior to administration of the compound deviatingfrom the normal.
 21. A compound according to claim 9, which is capableof decreasing the formation of interleukin 1 (IL-1), interleukin 6(IL-6), and/or tumour necrosis factor α (TNF-α), to afford decreasedproduction of nitric oxide and/or to downregulate the activity of nitricoxide synthase (NOS).
 22. A compound according to claim 9, which iscapable of stimulating the in vitro production of interleukin 8 (IL-8)and/or interleukin 10 (IL-10).
 23. An acid salt of any one of thecompounds of claim
 9. 24. A pharmaceutical composition comprising acompound according to claim 9 together with a pharmaceuticallyacceptable carrier.
 25. A compound according to claim 16, said haptenbeing 2,4-dinitrofluorobenzene (DNFB).
 26. A compound according to claim17, said hapten being 2,4-dinitrofluorobenzene (DNFB).
 27. A compoundaccording to claim 9, which ameliorates or inhibits formation of oedema,said oedema being associated with allergic reactions or inflammation.28. The compound of claim 1 which is capable of binding MC1 receptor invitro.
 29. The compound of claim 28 which is capable of activating MC1receptor in vitro.
 30. The compound of claim 28 which is capable ofblocking MC1 receptor in vitro.
 31. The compound of claim 1 which iscapable of stimulating second messenger cAMP in vitro.
 32. The compoundof claim 1 which is capable of inhibiting NO production in vitro. 33.The compound of claim 9 which is capable of binding MC1 receptor invitro.
 34. The compound of claim 9 which is capable of activating MC1receptor in vitro.
 35. The compound of claim 9 which is capable ofblocking MC1 receptor in vitro.
 36. The compound of claim 9 which iscapable of stimulating second messenger cAMP in vitro.
 37. The compoundof claim 9 which is capable of inhibiting NO production in vitro. 38.The compound of claim 9 which is capable of decreasing the formation ofinterleukin-1 (IL-1) in vitro.
 39. The compound of claim 9 which iscapable of decreasing the formation of interleukin-6 (IL-6) in vitro.40. The compound of claim 9 which is capable of decreasing the formationof tumor necrosis factor α (TNF-α) in vitro.
 41. The compound of claim 9which is capable of decreasing the formation of interleukin-1 (IL-1) invitro.
 42. The compound of claim 9 which is capable of downregulatingthe activity of nitric oxide synthase (NOS) in vitro.
 43. The compoundof claim 9 which is capable of decreasing the formation of tumornecrosis factor α (TNF-α).